Ochs Biochem

Biochemistry
Biochemistry
Second Edition
Raymond S. Ochs
Second edition published 2022
by CRC Press
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and by CRC Press

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© 2022 Raymond S. Ochs
First edition published by Jones and Bartlett Publishers, Inc 2012
CRC Press is an imprint of Taylor & Francis Group, LLC
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ISBN: 978-0-367-46553-7 (hbk)

ISBN: 978-0-367-46187-4 (pbk)
ISBN: 978-1-003-02964-9 (ebk)
Typeset in Warnock Pro

by Deanta Global Publishing Services, Chennai, India
To my wife, Jessica,
for her continued support.
Contents
Preface to the First Edition�� xvii

Preface to the Second Edition��xix
Acknowledgments��xxi
Author�� xxiii
Glossary��xxv
Chapter 1 Foundations��1
1.1 Origins of Biochemistry��1
1.2 Some Chemical Ideas��1
1.2.1 Reactions and Their Kinetic Description��2
1.2.1.1 Equilibrium��3
1.2.2 The Steady-State��5
1.3 Acid–Base Reactions��6
1.4 Redox��7
1.5 Energy��8
1.6 Cell Theory��9
1.7 Species Hierarchy and Evolution��11
1.8 Biological Systems��12
Key Terms�� 13
Bibliography�� 14
Chapter 2 Water��15
2.1 Structure of Water��15
2.1.1 Gas Phase Water��15
2.1.2 Partial Charges and Electronegativity��16
2.1.3 Condensed Phase Water: Hydrogen Bonding��18
2.1.4 Hydrogen Bonding in Condensed Phase Water�� 20
2.2 The Hydrophobic Effect��21
2.3 Molecules Soluble in Water�� 23
2.4 High Heat Retention: The Unusual Specific Heat of Liquid Water��24
2.5 Ionization of Water�� 25
2.6 Some Definitions for the Study of Acids and Bases�� 26
2.7 The pH Scale�� 27
2.8 The Henderson–Hasselbalch Equation�� 28
2.9 Titration and Buffering�� 30
Summary�� 31
Review Questions�� 31
Chapter 2 Addendum: The Dielectric�� 32
Key Terms�� 34
Chapter 3 Lipids�� 35
3.1 Significance�� 35
3.2 Fatty Acids�� 35
3.3 Triacylglycerols�� 39
3.4 Phospholipids��41
vii
viii Contents
3.5 Cholesterol�� 42
3.6 Lipid–Water Interactions of Amphipathic Molecules��44
3.7 Phospholipid Monolayers��47
3.8 Lipid Composition of Membranes�� 49
3.9 Water Permeability of Membranes and Osmosis�� 49
Summary�� 51
Chapter 3 Addendum: Inverted Micelles�� 52
Key Terms�� 53
Chapter 4 Carbohydrates�� 55
4.1 Monosaccharides�� 55
4.2 Ring Formation in Sugars��59
4.3 Disaccharides�� 62
4.4 Polysaccharides�� 64
4.4.1 Linear Polysaccharides�� 66
4.4.2 Branched Polysaccharides�� 68
4.5 Carbohydrate Derivatives�� 68
4.5.1 Simple Modifications�� 68
4.5.2 Substituted Carbohydrates�� 70
Summary�� 75
Chapter 4 Addendum: The Discovery of Stereoisomerism�� 76
Key Terms�� 77
Chapter 5 Amino Acids and Proteins�� 79

5.1 Common Structure of the Amino Acids�� 79
5.2 Biology of the Amino Acids�� 79
5.3 Amino Acid Individuality: The R Groups�� 80
5.3.1 Polarities�� 80
5.3.2 Functional Groups��81
5.4 Acid–Base Properties and Charge�� 85
5.4.1 Titration and Net Charge�� 85
5.4.2 Interactions between the α-Carboxylate and α-Amine Groups�� 87
5.4.2.1 Inductive Effect�� 87
5.4.2.2 Electric Field Effect�� 88
5.4.3 Multiple Dissociable Groups�� 90
5.5 The Peptide Bond�� 90
5.6 Peptides and Proteins�� 92
5.7 Levels of Protein Structure�� 93
5.7.1 Primary Structure�� 93
5.7.2 Secondary Structure�� 93
5.7.2.1 α-Helix�� 94
5.7.2.2 β-Sheet�� 94
5.7.3 Domains�� 94
5.7.4 Tertiary Structure�� 97
5.7.5 Quaternary Structure�� 98
5.8 Protein Folding�� 99
5.9 Oxygen Binding in Myoglobin and Hemoglobin��100
5.10 Other Binding Reactions Involving Proteins��103
5.10.1 Extracellular Binding Proteins��104
5.10.2 Cell Surface Binding Proteins��104
5.10.3 Intracellular Binding Proteins��104
Contents ix
5.11 Protein Purification and Analysis��105

5.11.1 Purification��105
5.11.2 Analysis��107
Summary�� 107
Chapter 5 Addendum: Atomic Charged Forms�� 108
Key Terms�� 109
Chapter 6 Enzymes��111
6.1 Energetics of Enzyme-Catalyzed Reactions��111
6.2 The Enzyme Assay and Initial Velocity��113
6.3 A Simple Kinetic Mechanism�� 114
6.3.1 Assumptions��115
6.3.2 The Michaelis–Menten Equation��115
6.4 How the Michaelis–Menten Equation Describes Enzyme Behavior��117
6.5 The Meaning of Km��119
6.6 Reversible Inhibition��120
6.6.1 Competitive Inhibition�� 121
6.6.2 Anticompetitive Inhibition (Uncompetitive)��122
6.6.3 Mixed Inhibition (Noncompetitive)�� 124
6.7 Double-Reciprocal or Lineweaver–Burk Plot��125
6.8 Allosteric Enzymes��126
6.9 Irreversible Inhibition��128
6.10 Enzyme Mechanisms��129
6.10.1 Nucleophilic Substitution��129
6.10.2 Acid–Base Catalysis�� 131
6.11 Enzyme Categories�� 132
6.12 Enzyme-Like Qualities of Membrane Transport Proteins�� 132
Summary�� 133
Chapter 6 Addendum: The Haldane Relationship�� 135
Key Terms�� 137
Chapter 7 Coenzymes��139
7.1 Coenzymes: Bound and Mobile��139
7.2 Coenzymes and Vitamins��140
7.3 Redox Coenzymes ��140
7.3.1 Nicotinamides��140
7.3.2 Ubiquinone ��144
7.3.3 Flavin Coenzymes��144
7.4 Acyl Transfers ��146
7.4.1 CoA��146
7.4.2 Carnitine��147
7.4.3 Lipoic Acid��148
7.5 Carboxylation��148
7.5.1 Biotin��149
7.5.2 Vitamin K��150
7.6 Exchange Coenzymes�� 151
7.6.1 Thiamine Pyrophosphate�� 151
7.6.2 Pyridoxal Phosphate ��152
7.7 Metal Ion Cofactors��154
7.7.1 Mg2+�� 155
7.7.2 Iron��155
x Contents
7.7.3 Copper��155
7.7.4 Co2+�� 155
7.7.5 Mn2+and Zn2+��156
7.7.6 Selenium��156
7.7.7 Dietary Essentials��156
Chapter 7 Addendum: The Vitamin K Cycle�� 157
Key Terms�� 158
Chapter 8 Metabolism and Energy�� 161

8.1 Origins of Thermodynamics�� 161
8.2 First Law of Thermodynamics�� 161
8.2.1 Heat and Work�� 161
8.2.2 Enthalpy��163
8.3 Entropy and the Second Law of Thermodynamics��164
8.3.1 Entropy as a Ratio of Heat to Temperature��165
8.3.2 Entropy as a Statistical Distribution of States��165
8.4 Free Energy��165
8.5 Standard Free Energy��167
8.6 Nonstandard Free Energy Changes��168
8.7 Near-Equilibrium and Metabolically Irreversible Reactions��169
8.8 ATP��170
8.9 Energy Coupling with ATP�� 171
8.9.1 Creatine Phosphokinase��173
8.9.2 NDP Kinase��173
8.9.3 Adenylate Kinase�� 174
8.10 Energy of Redox Reactions�� 174
8.10.1 Electricity Fundamentals�� 175
8.10.2 The Electrochemical Cell�� 176
8.10.3 Standard Reduction Potentials��177
8.10.4 Reduction Potentials and Free Energy��178
8.10.5 Reduction Potentials ��179
8.11 Mobile Cofactors and the Pathway View ��180
Summary�� 181
Chapter 8 Addendum: Free Energy Derivation�� 182
Key Terms�� 183
Chapter 9 Glycolysis��185
9.1 Glucose Transport��185
9.2 From Glucose to Pyruvate��186
9.2.1 Hexokinase��186
9.2.2 Glucose Phosphate Isomerase��189
9.2.3 Phosphofructokinase��190
9.2.4 Aldolase��193
9.2.5 Triose Phosphate Isomerase��194
9.2.6 Glyceraldehyde Phosphate Dehydrogenase��194
9.2.7 Phosphoglycerate Kinase��195
9.2.8 Phosphoglycerate Mutase��195
9.2.9 Enolase��196
9.2.10 Pyruvate Kinase��197
Contents xi
9.3 Completing the Pathway��199

9.3.1 Lactate Formation�� 200
9.3.2 Ethanol Formation�� 200
9.4 Energetics of Glycolysis��201
9.4.1 Pathway Thermodynamics��201
9.4.2 Red Blood Cell Shunt Pathway��203
9.4.3 Arsenate Poisoning��203
9.4.4 Fructose Metabolism��205
9.4.5 Energy Balance and Glycolytic Connections��206
9.5 Metabolic Connections to Glycolysis��207
9.5.1 Alternative Entry Points��207
9.5.2 Glycolytic Intermediates as Intersection Points��207
9.5.3 Alternative Endpoints of Glycolysis��208
Summary�� 208
Chapter 9 Addendum: Alternatives to Glycolysis�� 209
Key Terms�� 209
Chapter 10 The Krebs Cycle�� 211

10.1 A Cyclic Pathway�� 211
10.2 Acetyl-CoA: Substrate of the Krebs Cycle�� 212
10.3 Overview of Carbon Flow��216
10.4 Steps of the Pathway�� 217
10.4.1 Citrate Synthase�� 217
10.4.2 Aconitase��218
10.4.3 Cis-Aconitate as a Krebs Cycle Intermediate��220
10.4.4 Prochirality��220
10.4.5 The R/S System of Nomenclature��221
10.4.6 Fluoroacetate Poisoning��223
10.4.7 Isocitrate DH��223
10.4.8 α-Ketoglutarate DH Complex��224
10.4.9 Succinyl-CoA Synthetase��224
10.4.10 Succinate DH��225
10.4.11 Fumarase��226
10.4.12 Malate DH��226
10.5 Energy Balance��227
10.6 Regulation��228
10.7 Krebs Cycle as a Second Crossroad of Metabolic Pathways��229
Summary�� 230
Chapter 10 Addendum: Cyclic Pathways Connected to the Krebs Cycle�� 230
1. Glyoxylate Cycle��231
2. Itaconate Pathway��231
Key Terms�� 232
Chapter 11 Oxidative Phosphorylation��235

11.1 The Phenomenon��235
11.2 Mitochondrial Inner Membrane��236
11.3 Carriers of Electrons, Protons, or Both��237
11.3.1 Carriers of Electrons��237
11.3.2 Carriers of Protons��237
11.3.3 Carriers of Both Electrons and Protons��238
xii Contents
11.4 Membrane-Bound Complexes��238

11.5 The Electrochemical Cell and the Mitochondria��239
11.6 Electron Pathways��240
11.6.1 Sequence of Electron Flow��240
11.6.2 Energetics of Electron Flow�� 241
11.7 Mechanisms of the Mitochondrial Membrane Protein Complexes��243
11.7.1 Complex I: Proton Pump��244
11.7.2 Complex II: Succinate Dehydrogenase��245
11.7.3 Complex III: Loop Mechanism��245
11.7.4 Complex IV: Pump and Annihilation��247
11.7.5 Complex V: ATP Synthesis��247
11.7.5.1 The Stator��250
11.7.5.2 The Rotor��250
11.8 Proton-Motive Force��250
11.8.1 Electric Circuit Analogy to the Proton Gradient��250
11.8.2 Utilization of Δp beyond ATP Synthesis��251
11.9 Mitochondrial Membrane Transport��252
11.9.1 Adenine Nucleotide Translocase��252
11.9.2 Phosphate Exchange��252
11.9.3 Other Transport Proteins��252
11.9.4 Coupling of Oxidation and Phosphorylation��253
11.10 Uncoupling�� 254
11.10.1 Physiological Uncoupling: Brown Fat��256
11.11 Superoxide Formation by Mitochondria��256
11.12 Control of Mitochondria��257
11.13 How Mitochondria Can Utilize Cytosolic NADH��258
11.13.1 Glycerol Phosphate Shuttle��258
11.13.2 Malate/Aspartate Shuttle��258
Summary�� 260
Chapter 11 Addendum: Supercomplexes�� 261
Key Terms�� 262
Chapter 12 Photosynthesis��265
12.1 Light and Carbon Reactions��265
12.2 Chloroplasts��266
12.2.1 Orientations: N and P Sides of the Membrane��266
12.2.2 Light Reactions of Photosynthesis as a Reversed Oxidative Phosphorylation��267
12.2.3 Carbon Reactions of Photosynthesis: CO2 Fixation��268
12.3 Harnessing Light Energy��269
12.3.1 Light Absorption and the Antennae��270
12.3.2 Electron Transfer at Reaction Centers��270
12.4 Proton and Electron Flow for the Light Reactions��272
12.5 Cyclic Electron Transfer and Other Variations��274
12.6 The Calvin Cycle��274
12.6.1 Ribulose Bisphosphate Carboxylase��275
12.6.2 Reaction Steps Following Carbon Fixation to Glyceraldehyde-P: Energy
Consuming Portion��277
12.6.3 From GAP to the RuBP: Overview��277
12.6.4 From GAP to the RuBisCo Step: Reactions��278
12.7 Variations in CO2 Handling: C3, C4, and CAM Plants��282
12.8 Pathway Endpoints: Sucrose and Starch�� 284
Summary�� 285
Contents xiii
Chapter 12 Addendum: Dark vs Carbon Reactions�� 286

Key Terms�� 286
Chapter 13 Carbohydrate Pathways Related to Glycolysis��289

13.1 Glycogen Metabolism��289
13.1.1 Glycogen Synthesis��289
13.1.2 Glycogenolysis��291
13.1.3 Physiological Context of Glycogen Metabolism��296
13.1.4 Regulation of Glycogen Metabolism by Glucagon��296
13.1.5 Regulation of Glycogen Metabolism by Epinephrine��299
13.1.6 Regulation of Glycogen Metabolism by Insulin�� 300
13.1.7 Regulation of Glycogen Metabolism by AMP Kinase��303
13.2 Gluconeogenesis�� 304
13.2.1 Lactate Dehydrogenase as a Gluconeogenic Enzyme�� 304
13.2.2 Pyruvate to PEP��305
13.2.3 Indirect Transport of Oxaloacetate from Mitochondria to Cytosol�� 306
13.2.4 Fructose-1,6-P2 to Fructose-6-P��309
13.2.5 Glucose-6-P to Glucose�� 310
13.2.6 Pathway Integration: Glycolysis, Glycogen Metabolism, and Gluconeogenesis�� 311
13.2.6.1 The Feeding–Fasting Transition�� 311
13.2.6.2 The Resting–Exercise Transition�� 312
13.3 The Pentose Phosphate Shunt�� 313
13.3.1 Oxidative Stage�� 314
13.3.2 Nonoxidative Stage�� 314
13.3.2.1 Reactions of the Nonoxidative Stage�� 315
13.3.2.2 A Pseudo Three-Dimensional View and Comparison to the Calvin Cycle�� 316
13.3.3 Distribution Between NADPH Production and Ribose-5-P�� 318
13.4 Galactose Utilization�� 318
Summary�� 320
Chapter 13 Addendum: Phosphorylation of Glycogen�� 321
Key Terms�� 322
Chapter 14 Lipid Metabolism��325

14.1 Absorption of Dietary Lipids��325
14.2 Fatty Acid Oxidation��326
14.2.1 Activation��327
14.2.2 Transport��328
14.2.3 β-Oxidation��330
14.2.3.1 Unsaturated Fatty Acids��331
14.2.3.2 Odd Carbon-Numbered Fatty Acids��331
14.2.3.3 Shorter and Longer Fatty Acids��334
14.3 Ketone Body Metabolism��334
14.4 Fatty Acid Biosynthesis��335
14.4.1 Export of Acetyl-CoA to the Cytosol��336
14.4.2 Carboxylation of Acetyl-CoA to Malonyl-CoA��339
14.4.3 Sequential Addition of Two-Carbon Fragments to Form Palmitate�� 340
14.4.3.1 Loading��341
14.4.3.2 Condensation��341
14.4.3.3 Reduction��342
14.4.3.4 Dehydration��343
14.4.3.5 Second Reduction��343
xiv Contents
14.5 Triacylglycerol Formation��343

14.6 Phospholipid Metabolism��343
14.7 Cholesterol Metabolism�� 346
14.8 Other Lipids��351
14.8.1 Eicosanoids��351
14.8.2 Sphingolipids��353
14.8.3 Unusual Bacterial Fatty Acids��353
14.9 Overview of Lipid Metabolism in the Fed and Fasted States��353
14.10 Integration of Lipid and Carbohydrate Metabolism��355
14.10.1 Lipid and Carbohydrate Intersections in the Feeding–Fasting Transition��357
14.10.2 Lipid and Carbohydrate Intersections in the Resting–Exercise Transition��357
14.10.3 Lipid and Carbohydrate Intersections in Diabetes Mellitus��357
Summary�� 358
Chapter 14 Addendum: The Isoprenes�� 359
Key Terms�� 360
Chapter 15 Nitrogen Metabolism��363

15.1 The Nitrogen Cycle��363
15.2 Reaction Types in NH3 Assimilation�� 364
15.2.1 Redox-Neutral�� 364
15.2.2 Redox-Active�� 364
15.2.3 Redox-Balanced��365
15.3 Metabolically Irreversible Nitrogen Exchange Reactions��365
15.4 Near-Equilibrium Nitrogen Exchange Reactions�� 366
15.4.1 Glutamate DH�� 366
15.4.2 Transaminases�� 366
15.5 The Urea Cycle�� 368
15.5.1 [NH3] Exceeds [Aspartate]�� 368
15.5.2 [Aspartate] Exceeds [NH3]��369
15.5.3 Steps from NH3 to Citrulline��369
15.5.4 Cytosolic Steps of the Urea Cycle��369
15.5.5 Overall Urea Cycle��371
15.6 Amino Acid Metabolism: Catabolism��372
15.6.1 Branched-Chain Amino Acid Breakdown��372
15.6.2 Threonine��372
15.6.3 Lysine��372
15.6.4 Tryptophan��374
15.6.5 Phenylalanine and Tyrosine Degradation��374
15.6.6 Amino Acids Directly Connected to Major Metabolic Pathways��376
15.6.7 Arginine, Proline, and Histidine Are All Converted to Glutamate��377
15.6.8 One-Carbon (1C) Metabolism and Serine, Glycine, and Methionine Breakdown��378
15.7 Amino Acids: Anabolism��380
15.7.1 Nonessential Amino Acids��382
15.7.2 Essential Amino Acids��382
15.7.3 Aromatic Amino Acid Biosynthesis��382
15.8 Nucleotide Metabolism��383
15.8.1 Pyrimidine Synthesis�� 384
15.8.2 Pyrimidine Degradation��386
15.8.3 Purine Synthesis��386
15.8.4 Purine Degradation��391
15.8.5 Salvage Reactions��393
15.8.6 Purine Nucleotide Regulation��393
Contents xv
15.8.7 Purine Nucleotide Cycle��394

15.8.8 Deoxynucleotide Formation��394
15.8.9 A Unique Methylation to Form dTMP��395
15.9 Other Nitrogen Pathways��398
Summary�� 399
Chapter 15 Addendum: Nitrogen Disposal�� 400
The Tilapia of Lake Magadi�� 401
The Alligator and the Crocodile�� 401
Low Creatinine and Liver Failure�� 401
Key Terms�� 402
References�� 403
Chapter 16 Nucleic Acids�� 405

16.1 Strand Structures of the Nucleic Acids�� 405
16.2 Structure of the Double Helix��407
16.3 Supercoiling�� 410
16.4 Histones��411
16.5 Replication�� 412
16.5.1 Initiation�� 412
16.5.2 Replication Fork and the Replisome�� 413
16.5.3 Primer Formation�� 414
16.5.4 Creating the Double Helix�� 414
16.5.5 Distinctive Features of Eukaryotic Replication�� 415
16.6 DNA Repair�� 415
16.6.1 Mismatch Repair�� 415
16.6.2 Excision Repair�� 416
16.6.3 PARP�� 416
16.7 Transcription�� 416
16.7.1 RNA Polymerase Binding to DNA�� 418
16.7.2 Transcription Events in E. coli�� 419
16.7.3 Eukaryotic Transcription�� 419
Summary�� 423
Chapter 16 Addendum: Restriction Enzymes and PCR �� 425
Restriction Enzymes�� 425
PCR�� 425
Key Terms�� 426
Chapter 17 Protein Synthesis and Degradation��429

17.1 Three Forms of RNA Employed in Protein Synthesis��429
17.1.1 tRNA��429
17.1.2 rRNA and the Ribosomes��431
17.2 The Genetic Code��432
17.3 Steps in Protein Synthesis��433
17.3.1 Initiation��433
17.3.2 Elongation��433
17.3.3 Termination��436
17.3.4 Distinctive Features of Eukaryotic Translation��437
17.3.5 Regulation of Eukaryotic Translation��437
17.4 Co-Translational and Post-Translational Modifications of Proteins��437
17.4.1 Co-Translational Modifications��437
17.4.2 Post-Translational Modifications�� 440
xvi Contents
17.5 Protein Degradation�� 440

17.5.1 Extracellular Proteases�� 440
17.5.2 Intracellular Proteases��442
17.6 The mTOR Pathway in the Control of Protein Synthesis��443
Summary�� 445
Chapter 17 Addendum: Skeletal Muscle and Exercise�� 446
Key Terms�� 446
Appendix�� 449
Index��461
Preface to the First Edition
CONSIDERING METABOLISM ORGANIZATION
Metabolic diseases, such as diabetes and others associ- The presentation of biochemistry topics is mostly a classi-
ated with the current obesity crisis, have thrust metab- cal one, with a slight divergence: the introduction to lipids
olism into the forefront of popular thinking. In many directly follows the chemistry of water. This is in keeping
ways, metabolism is the central science of biochemistry. with presenting molecular classes in the order of increas-
This is the view I have adopted as the core concept of this ing chemical complexity: water, lipids, carbohydrates,
textbook. nitrogen compounds. It also has the virtue of contrast-
Having a research background in metabolism, a ing water solubility with water insolubility in the case of
long-standing interest in the fundamental topics of lipids.
biochemistry – including kinetics and thermodynamics – When using this book for medically oriented courses,
and having taught a one-semester biochemistry course the chapter on photosynthesis may be omitted. In that
for over 25 years, I have long wanted to write a book that case, however, the photosynthetic “dark reactions” should
reflects the whole of the subject with the unifying theme at least be referenced, as they are similar to those in the
of metabolism. pentose cycle; this analogy is made explicit in the chapter
on carbohydrate pathways.
BREVITY
KEY FEATURES
A key consideration when writing this text was to keep
the book short enough and approachable enough that ◾◾ Dual diagrams of enzymatic reactions. A unique
a student can read it in one semester. The alternative method of presenting electron flow for reac-
approach – having a book far too long for continuous tion mechanisms was devised for this book.
reading and having the instructor suggest which portions For each mechanism, the substrate, intermedi-
to omit – is already well represented. In my experience, for ates, and product are presented on the top line.
students who are not biochemistry majors, the fragmenta- Below a separator (dotted line), the molecules
tion resulting from parsing longer texts leads to less read- are redrawn with their electron flows, using the
ing and, therefore, less understanding. The areas of special traditional curved arrows. This separation allows
interest to the instructor can be readily augmented with the student to visualize the result of the elec-
primary sources, while the textbook provides continuity tron flow, which is commonly obscured by the
and context. need to show the electron arrows for the next
I have emphasized recurring ideas such as commonal- transformation.
ities in chemical reaction mechanisms and pathway con- ◾◾ Word Origins feature box. Included in most
struction as much as possible. The decision of whether chapters is a box that provides a short history of
the book is for teaching or reference is decidedly in favor certain words that are rich in meaning, without
of teaching; for example, only a few protein domains which it is often more difficult to understand the
are presented. The wealth of information available on underlying concept. Providing an explanation
the Web (notably ncbi.nlm.nih.gov) can substitute for of their origins helps students become familiar
a more extensive collection. The present text will pro- with these important terms and achieve a better
vide students with an introduction to the foundations of understanding of what is being described.
biochemistry. ◾◾ Thermodynamics treatment. The development of
thermodynamics for metabolic purposes leads to
xvii
xviii Preface to the First Edition
the distinction between two classes of enzymes: often, "K M " becomes a focal point and is treated
near-equilibrium and metabolically irreversible. as an equilibrium constant rather than a steady-
This allows a simplification, as near-equilibrium state constant. This common misuse of K M versus
reactions are not sites of cellular regulation. The Vmax/K M is part of the reason that many students
roles of standard, actual, and near-equilibrium have difficulty understanding enzyme kinetics,
states for free energy are distinct and consistently and it is a problem this text carefully avoids.
presented to aid student understanding. ◾◾ Minimalist molecular biology treatment. The
◾◾ Chemical mechanisms. A study of biochemistry essence of molecular biology is included in the
should impart a viewpoint enriched by under- final two chapters. The emphasis is on provid-
standing the underlying chemistry of events in ing an overview with a chemical perspective. For
living systems. This extended view of biology is example, stacking interactions in DNA, the basis
best achieved by understanding how enzymatic for forming the double helix, are explained in
reactions function. All of the background chem- simple, chemical terms.
istry needed for this text should be covered by ◾◾ The pathway view. The distinction between
prerequisite courses of chemistry. Some fur- a reaction view and a pathway view is clearly
ther information is presented in the appendix; emphasized to facilitate student comprehension.
a review of organic chemistry reaction mecha- For example, the distinction between a bound
nisms (most critically, nucleophilic reactions) cofactor like FADH2 from a free cofactor like
may be necessary for those who are less comfort- NADH becomes obvious: only NADH can trans-
able with the material. fer electrons between metabolic reactions.
◾◾ Enzyme kinetics treatment. The text emphasizes ◾◾ Appendix. The appendix of this text contains
direct plots of substrate concentration against both entries for students needing a little extra
initial velocity, introducing double-reciprocals help and more advanced material that, while
only after a complete development of the subject. perhaps not appropriate for the level of this
While in widespread use, the double-reciprocal particular text, is nonetheless important to the
form is difficult to visualize and leads to the field of biochemistry as a whole. For remedia-
false impression that memorizing patterns of tion, basic ideas of mathematics and chemistry
lines for enzyme inhibition provides insight into with which students traditionally have difficulty
how inhibitors work. Instead, direct plots, with can be found in the appendix, as can certain
an emphasis on the behavior of reaction velocity extended pathways like amino acid and choles-
at different substrate concentrations, deliver the terol routes. The advanced material includes the
message clearly. Coupled with everyday descrip- mechanism of aconitase and the role of fluoroac-
tions of the different types of inhibition, these etate, which represent milestone achievements
critical ideas are easily grasped. A further dis- in biochemistry. These may be of interest to the
tinct notion is the use of the kinetic term Vmax more inquisitive student wishing to go beyond
/K M rather than K M in developing kinetics. Too the fundamentals.
Preface to the Second Edition
Metabolism remains the focal point of the textbook, as linear or cyclic: repetition-variation. This description
does the goal of maintaining a one-semester book. The fits several pathways and provides a new way of thinking
organization is the same, although a new chapter on coen- about them. The second, introduced in the final chap-
zymes follows enzymes. This chapter can either be read in ter, is pathway-reversible versus pathway-irreversible as
sequence to expand the chemistry of enzymes or skimmed a means to discriminate between regulation that can be
to consult when specific coenzymes are encountered later. reverted by opposing reactions (reversible) or incorpo-
Some expansion of chemistry is provided, such as a rated into proteins for their entire existence (irreversible).
more detailed treatment of acid–base and redox reactions I have tried to make more explicit in this edition the
and a fuller explanation of prochirality, as my own under- underpinnings of chemistry in biological sciences. In the
standing of that concept has evolved. Each chapter has a present climate, there is a tendency for students to believe
new addendum, providing an optional reading topic, usu- that they can look up whatever they need rather than
ally of a more advanced nature than the book narrative. examine the roots of the subject. This view is unfortunate;
The organizing principles of near-equilibrium and it means that every new fact or idea is searched anew and
metabolically irreversible enzymes are carried over from committed to memory in isolation. A few principles of
the first edition. In this edition, I have added two more. biochemistry can be instead the foundation of a deeper
The first is an idea taken from the advertising industry, understanding.
applied to perplexing pathway routes that are not quite
xix
Acknowledgments
The invaluable feedback from reviewers is gratefully David Hilmey, St. Bonaventure University
acknowledged: Blaine Legaree, Keyano College
Lisa Lindert, California State University - Sacramento
Sergio Abreu, Fordham University Meagan Mann, Austin Peay State University
Lois Bartsch, Graceland University Nick Menhart, Illinois Institute of Technology
Sajid Bashir, Texas A&M University - Kingsville Abdel Omri, Laurentian University
Mrinal Bhartacharjee, Long Island University Gordon Rule, Carnegie Mellon University
Debra Boyd-Kimball, University of Mount Union Mary Railing, Wheeling Jesuit University
Barbara Bowman, University of California - Berkeley Gerald Reeck, Kansas State University
Jeanne Buccigross, College of Mount St. Joseph Abbey Rosen, Marian College of Fond du Lac
Mickael Cariveau, Mount Olive College Frank Schmidt, University of Missouri
John Brian Chapman, Federation University Michael Sehorn, Clemson University
Australia Kavita Shah, Purdue University
James Cheetham, Carleton University Andrew Shiemke, West Virginia University
Zhe-Sheng Chen, St. John’s University Amrurhesh Shivachar, Texas Southern University
David Eldridge, Baylor University Todd Silverstein, Willamette University
Susan Evans, Ohio University Madhavan Soundararajan, University of
John Fain, University of Tennessee Health Science Nebraska - Lincoln
Center Salvarore Sparace, Clemson University
Sue Ford, St. John’s University Vicky Valancius-Mangel, Governors State University
Matthew Gage, Northern Arizona University David Watt, University of Kentucky
Eric Gauthier, Laurentian University Wu Xu, University of Louisiana - Lafayette
Neil Haave, University of Alberta - Augustana Mali Yin, Sarah Lawrence College
Campus
xxi
Author
Raymond S. Ochs is a biochemist with a career-long concern major pathways of liver and muscle, includ-
specialty in metabolism spanning 30 years. Previously, ing glycolysis, gluconeogenesis, ureogenesis, fatty
he has written the textbook Biochemistry, the mono- acid metabolism, glycogen metabolism, and control
graph Metabolic Structure and Regulation, contributed by cAMP, Ca 2+, diacylglycerol, and AMPK. He is cur-
the metabolism chapters to another text, Principles of rently professor of pharmacy at St. John’s University
Biochemistry, and co-edited a collection of articles pub- in New York, teaching biochemistry, physiology, and
lished as Metabolic Regulation. His research interests medicinal chemistry.
xxiii
Glossary
A allopurinol – A synthetic drug (a hypoxanthine analog)

used to treat gout through its inhibition of xan-
α-carbon – Carbon adjacent to a carboxyl group; often thine oxidase and thus uric acid formation.
referred to as the carbon bearing a carboxyl allosteric – Literally “other site”, a binding site for a pro-
group and an amino group in an amino acid. tein apart from the active site for binding or
α-hydrogen – The hydrogen attached to the α-carbon of catalysis, involved in regulation.
an amino acid. alpha (form) – The hydroxyl group of an anomeric car-
ACP-SH – See acyl carrier protein. bon that is on the same side of the ring as the
acceptor site – During protein synthesis, the location of hydroxyl group that determines the d-form of
the ribosomal binding site for aminoacyl-transfer most monosaccharides.
RNA (also termed A site). amide – A bond formed (formally by dehydration)
acceptor stem – Located at the opposite end of the anti- between an amine and a carboxyl group.
codon on a transfer RNA, the 3' end with a CCA amino acids – The building blocks of proteins and inter-
sequence, where the appropriate amino acid mediates in metabolism, all contain an amine
bonds with the terminal end adenine (A) residue. group and a carboxyl group attached to the
activation energy – Energy beyond the difference in α-carbon.
energies between substrates and products neces- amino-acyl-tRNA synthetases – Enzymes that selec-
sary to bring about reaction. tively recognize and bind an amino acid to a
active site – Specific region of an enzyme where sub- matching transfer RNA.
strates bind and reaction catalysis takes place. amino end – The end of a protein or peptide that termi-
acyl carrier protein – A small protein containing a nates in a free α-amine (also, the N end).
free –SH from a cysteine residue that forms ampere – A unit of measure for electrical current.
thioester intermediates in fatty acid biosynthesis. amphipathic – Having both polar and nonpolar parts
adipocyte – Fat cell. within a single molecule. Example: fatty acids.
adrenergic receptors – Hormone receptors for epineph- ammoniotelic – Organisms that produce ammonia as
rine or norepinephrine (also known as adrenaline their major nitrogen excretion product.
and noradrenaline, respectively). anabolic steroid – A synthetic hormone that promotes
affinity – Binding ability, characterized by an equilibrium protein synthesis and cell growth, sometimes
constant. used by athletes to increase muscle size and
affinity chromatography – A form of chromatography strength.
in which the stationary phase contains molecules anapleurotic – Filling reaction for a cyclic pathway. For
that selectively combine with components of the example, pyruvate carboxylase catalyzes oxalace-
mobile phase. tate formation, an intermediate of the Krebs cycle.
agar – A mixture of agarose and agaropectin with a gelat- annihilation – A mechanism of formation of the mito-
inous consistency that can support growth of chondrial proton gradient in which proteins are
microorganisms for in vitro analysis. removed from the matrix space through their
agaropectin – A polysaccharide similar to agarose, reaction with oxygen to form water.
except that it has branched sugars and sulfuric anomeric carbon – In a sugar ring, the carbon bearing
acid esters to some of its hydroxyl groups. the hydroxyl group that was a carbonyl in the
agarose – A polysaccharide found in certain seaweeds, it open chain form.
has alternating galactosyl and anhydro-L-gluco- antennae – In photosynthesis, a large pool of chlorophyll
syl subunits. molecules that absorb light.
aldoses – Sugars containing an aldehyde (at carbon 1). anticodon – Located on one end of transfer RNA, a
alkaloids – A class of naturally occurring nitrogenous sequence of three adjacent nucleotides. Functions
organic compounds found in plants, some fungi, during the translation phase of protein synthesis
and animals. Examples: caffeine, morphine, nico- to bind to the complementary coding triplet of
tine, quinine, and strychnine. nucleotides in messenger RNA (see codon).
xxv
xxvi Glossary
anticompetitive inhibition – Occurs when an enzyme biopterin – A pterine (heterocycle) redox cofactor that
inhibitor only binds to an enzyme substrate (ES) can transfer single electrons as well as electron
complex (see uncompetitive inhibition). pairs.
antiparallel – In β-sheet of an amino acid sequence, biotin – A carboxylation cofactor in which an intermedi-
when the strands run in opposite directions. ate CO2 is transiently attached to a nitrogen atom
aquaporins – Protein channels selective for water of this molecule.
transport. bisphosphonate – A drug containing a phosphorous
apoprotein – The protein portion of a larger complex, atom with a P–C bond used in the treatment of
formed with a prosthetic group. osteoporosis, as it inhibits osteoclasts which dis-
apoptosis – Programmed cell death. This process pre- solve bone.
vents release of cellular components as a cell is Bohr effect – The diminished binding of oxygen to hemo-
extinguished either during development or in globin in response to an increased concentration
response to some disease states. of protons.
Arrhenius definition – A classification of acid–base branch points – Location on a polymer chain in which a
reactions, for which an acid increases the proton chain diverges to form a new polymer end. This
concentration in water, and a base decreases the usually refers to polysaccharides, which have
proton concentration in water. multiple hydroxyl groups, in which the branch
assay – Quantitative or qualitative procedure to analyze points are often αl→6 linkages.
a substance. Bronsted definition – An acid–base definition, in which
assimilation – The incorporation of nutrients into liv- an acid is a proton donor, and a base is a proton
ing tissue. Also used specifically as a term for acceptor.
bacterial conversion of nitrate and nitrite into brown adipose – A type of fat cell with numerous
ammonia. small lipid droplets rather than just one, many
autophagy – Lysosomal digestion of a cell's own cyto- mitochondria, and an uncoupling protein that
plasmic material. can initiate uncoupled respiration to generate
auxotrophic – Organisms requiring carbon sources heat.
other than CO2. buffering region – The region of a titration curve in which
addition of acid or base has minimal effects on
the pH; visually, a flat region in the the curve.
B bundle sheath – Inner plant cells that converts the four-
carbon malate to pyruvate and CO2.
:B – Enzyme-bound base.
β-barrel – Portion of a protein domain formed from mul-
tiple β-sheets. C
beta (form) – The hydroxyl group of an anomeric carbon
that is on the opposite side of the ring from the C3 plants – Plants that directly incorporate CO2 into
hydroxyl group that determines the d-form of P-glycerate (the C3) rather than intermediate
most monosaccharides. conversion to a C4 for the purpose of localizing
β-oxidation – The process of oxidative degradation of CO2.
fatty acids, where two-carbon units are sequen- C4 plants – Plants with specialized pathways to protect
tially removed from the carboxyl end of the against RuBisCo using O2 as an alternative sub-
polymer. strate to CO2. The C4 refers to a four-carbon
bile – A mixture of bile salts, phospholipids, and choles- intermediate that is synthesized from PEP, carry-
terol that is secreted by the liver, stored in the ing CO2 as the fourth carbon and releasing it in a
gallbladder, and released into the duodenum, separate inner cell.
where it forms mixed micelles with dietary lipids calmodulin – A small calcium-binding protein attached
to facilitate their hydrolysis. to some proteins noncovalently to confer calcium
bile salts – Modified cholesterol derivatives that are sensitivity.
amphipathic and can form mixed micelles with Calvin cycle – Cyclic pathway in photosynthetic organ-
dietary lipids, such as cholate. isms that serves to convert three CO2 molecules
binding change – A mechanism for complex V to 3-P-glycerate.
(F1FoATPase), proposed by Paul Boyer, that a pro- CAM plants – Crassulacean acid metabolism. These
tein segment of the complex moves among the plants have a physical barrier modification to
three adenine nucleotide binding sites, driven by prevent O2 from reaction at RuBisCo. Pores open
proton entry. at night to admit CO2
Glossary xxvii
cap – A 7-methylguanosine in 5'–5' triphosphate linkage positive or negative, in which opposite charges
to the first nucleotide of the mRNA; this is added attract and like charges repel.
post transcriptionally and is not encoded in the charged tRNA – A tRNA with an amino acid attached
DNA. (tRNAamino acid).
capping – At the terminal point of a chromosome (see chemiosmotic hypothesis – The theory that protons are
telomere), structures protecting the ends from the essential intermediate between oxidation of
degradation and fusion, a requirement for linear substances (with attendant electron transfer) and
DNA. the phosphorylation of ADP to form ATP.
carbon cycle – Global reactions of CO2 between auxo- chelate – See dative bond.
trophs (such as animals) and photosynthetic chiral – An asymmetric carbon center in a molecule;
organisms (such as plants). Latin for handedness. A carbon atom to which
carbon fixation – Net incorporation of CO2 into sugars four distinct groups are attached is a chiral
and ultimately other carbon compounds in cells. center.
carbon reaction – Reactions in photosynthetic organ- chloroplast – Plant cellular organelle in which photosyn-
isms that convert CO2 into sugars. Formerly thesis occurs.
known as dark reactions. chorismate – An intermediate in the biosynthesis of aro-
carboxyl end – The end of a protein or peptide that ter- matic amino acids. The common pathway to aro-
minates in a free α-carboxyl group (also, the C matic amino acids bifurcates at chorismate: one
end). leads to phenylalanine and tyrosine; the other
carnitine shuttle – A pathway for transport of fatty acyl- leads to tryptophan.
CoA molecules into mitochondria that involves chromatography – A group of analytic techniques for
intermediate conversion of the thioester into a purification of materials that works by differ-
carnitine ester. ent molecular affinities within the mixture for a
catalytic rate constant – Rate constant for the spe- mobile phase and a stationary phase.
cies directly leading to product in an enzyme chylomicron – A lipoprotein particle carrying dietary
mechanism. In our simple enzyme mechanism, lipid synthesized in intestinal epithelial of the
that species is ES and the catalytic rate constant small intestine that is transported by the lym-
is kcat. phatic system to the blood and subsequently
catalytic triad – Three proximal amino acid residues in an various tissues in the body.
enzyme active site that are involved in catalysis, chyme – Liquefied digest following physical processing in
connected by a relay system that uses acid–base the stomach that exits through the pylorus to the
catalysis to enhance a nucleophile, commonly a duodenum for digestion.
serine residue. citric acid cycle – A cyclic series of reactions that cata-
cell theory – A concept that cells are the fundamental lyzes the complete oxidation of acetyl-CoA to
unit of living systems, arising from other cells. CO2 (see Krebs cycle, tricarboxylic acid cycle).
cellulose – A linear polymer of glucose that has β1→4 classical thermodynamics – Thermodynamic princi-
linkages and a very regular three-dimensional ples developed in the 19th century based upon
solution structure; an insoluble polysaccharide postulates (called “laws of thermodynamics”)
that comprises plant cell walls, vegetable tissue that have never been contradicted. The first law
fibers (e.g., cotton), and wood. Cellulose is the is energy conservation; the second postulates
most abundant macromolecule on earth. an increase in entropy (roughly, disorder) in the
ceramide – A structure that is an analog of diacylglyc- universe.
erol, except with no acyl groups; an intermediate CMC – see critical micelle concentration.
in the biosynthesis of sphingolipids. cobalamin – see Vitamin B12 .
change in state – Thermodynamic variables that are CoA – Coenzyme of acetylation. Its sulfhydryl group par-
defined by the difference between one condi- ticipates in exchange of acyl groups in metabolic
tion and another undergo a change in state if the reactions.
change is independent of the path taken between codon – A triplet of bases in the messenger RNA that,
the two, and leads to a difference in state vari- when bound to the ribosome, bind to the three
ables, such as entropy. bases of the anticodon region in transfer RNA
chaperones – A protein that assists folding of another (see anticodon).
protein into its active conformation, either dur- coenzyme – Molecules associating with enzymes that
ing the synthesis of the protein or afterwards. participate in the chemistry of the catalytic
charge – An electrical primitive term having no deeper reaction.
definition that is an entity considered either colloids – Early name for macromolecules.
xxviii Glossary
collision theory – A concept of reaction kinetics that the cosubstrate – A substrate of an enzymatic reaction that
rate of a reaction is proportional to the number is not part of the pathway view and connects with
of collisions. other reactions in the cell; it is a mobile cofactor.
Coulombs – Units of charge, based on current (rate of Cori cycle – An inter-organ metabolic pathway in which
charge) and time (seconds); the units are amp-sec. glucose forms lactate in the muscle (glycolysis),
competitive inhibition – A form of reversible inhibition and the lactate is converted to glucose in the liver
in which the inhibitor binds exclusively to the (gluconeogenesis).
free enzyme form. coupled – In oxidative phosphorylation, it refers to the
complementarity – The selective binding of nucleotides oxidation of substrates, usually through the
within a DNA or RNA chain to another nucleic intermediacy of NADH, to O2, tied to the pro-
acid that follows the rule that A binds T (or U, if duction of ATP.
the nucleic acid is RNA), and G binds C. coupling – see coupled.
complete pathway – A metabolic pathway that is bal- critical micelle concentration – The concentration
anced, so that all mobile cofactors are regenerated. of fatty acids (or other lipid molecules) added
concerted – Electron movements within a chemical to a water solution that enables them to form a
mechanism that occur at the same time; a single- micelle, so that further addition will not increase
step reaction. the free concentration. The concept is also applied
condensed phase – A form of matter in which the mole- to molecules that form non-micelle aggregates
cules (or atoms) are incompressible, such as a solid such as certain phospholipids.
or liquid. current – Rate of movement of charge, measured in amps.
conjugated – Electron clouds of π-bonds of at least two CRISPR – A technique for incorporating DNA segments
unsaturated centers are able to interact, enabling into organisms, derived from a defense system
delocalization and stabilization. Commonly formed by bacteria against bacteriophages. The
applies to double bonds separated by a single full name is “Clustered Regularly Interspaced
methylene moiety. Short Palindromic Repeats”.
conjugate acid – A positively-charged proton donor cyclooxygenase – An enzyme converting arachidonate
formed by protonating a neutral Bronsted base. to a cyclic lipid that is the first step in the syn-
conjugate base – A negatively-charged proton acceptor thesis of prostaglandins and prostacyclins. The
formed by dissociating a proton from a neutral enzyme is the target of aspirin inhibition.
Bronsted acid. cyclosporine – A cyclic peptide (1200 Da) that causes
consensus (sequences) – Similar sequences between inactivation of the protein phosphatase calcineu-
strands of DNA in different species that have a rin and suppresses formation of the cytokine IL-2
common function. The term can also be applied from T cells, leading to immunosuppression. The
to RNA or protein sequences. drug known as an immunophilin, used to pre-
coordinate covalent bond – See dative bond. vent organ rejection in transplant patients.
cooperativity – A model to explain deviations from the cytochrome P450 – Any of a group of enzymes catalyz-
normal Michaelis–Menten curve for enzyme ing mixed-function oxidation, leading to hydrox-
activity, or the S-shaped curve for binding oxy- ylation of xenobiotics, including drugs, which
gen to hemoglobin. The concept usually involves renders them more water soluble and facilitates
conformational changes of individual subunits in their excretion from the body.
a multi-subunit protein that subsequently alter the cytosol – The major water space within a cell.
affinity of other subunits for substrate or ligand.
coordination number – The number of directed molec-
ular bonds from electron-rich atoms to a metal D
like Fe2+.
core – After RNA is synthesized in transcription, the σ dark reactions – Earlier term for reactions of photosyn-
subunit of the holoenzyme dissociates, thereby thesis that do not require light, now replaced by
producing the core complex, α2ββ’. carbon reactions.
coproduct – A product of an enzymatic reaction that is dative bond – A coordinate-covalent, or chelate bond,
not part of the pathway view and connects with where electrons are not shared, and yet there is
other reactions in the cell; it is a mobile cofactor. directionality to the bond.
corrosive – A gas that is reactive; the term refers to the dehydrogenases – A category of enzymes that catalyzes
ability to oxidize metal. Oxygen is an example of the exchange of hydrides between substrates and
a corrosive gas. nicotinamide nucleotides (NAD+ and NADP+).
Glossary xxix
denitrification – Process of removing nitrogen or nitro- electrochemical cell – A device to measure redox poten-
gen groups from a compound or reducing nitrites tials by separating oxidation from reduction in
and nitrates to N2. separate half-cells.
dielectric constant – A number assigned to a molecule electrical potential – A difference in charge between
that measures how well it separate ions in solu- two points; also known as a voltage.
tion. Values are based on a vacuum, set at 1.0; electrode – A metal that serves to enable a redox reaction
water has a value of about 80. on its surface and carry electrons to or from a
diester – A double ester; for example, a phosphate group wire in an electrochemical cell.
esterified both to choline and to glycerol in phos- electrogenic – An exchange process for molecules
phatidyl choline. across a biological membrane that is not charge
diffusion – Random motion of molecules that results in balanced.
their redistribution from regions of high to low electron-releasing – A through-bond effect of donating
concentration. electrons due to a difference in the electronega-
dihydrobiopterin – A redox cofactor with electron tivity between atoms across a bond.
transfer properties similar to flavins. electron-withdrawing – A through-bond effect of
dinuclear copper center – Redox transferring copper accepting electrons due to a difference in the
metal ion complex found in cytochrome oxidase. electronegativity between atoms across a bond.
dipole moment – A measure of bond polarity, it deter- electron paramagnetic resonance – A type of spec-
mines electron distribution across a bond. The troscopy in which there is resonant absorption
value is greater if the atoms across the bond have of radiation from chemical substances with
greater differences in electronegativity. unpaired electrons. A spectrographic technique
disaccharide – Two sugars linked by a glycosidic bond. that identifies and measures free radicals.
DNA gyrase – A topoisomerase, involved in unwinding electron sink – An electron deficient portion of a mol-
coiled DNA. ecule that attracts electrons in the course of a
DNA ligase – An enzyme that catalyzes diester forma- reaction mechanism.
tion between adjacent nucleotides in double- electron transport chain – A connected series of redox
stranded DNA. reactions that exist in the mitochondrial mem-
DNA polymerase – Enzymes active in the replication brane that accept electrons from NADH and
and repair of DNA that catalyze the synthesis of a donate them to O2. A similar structure exists in
new DNA strand from an existing DNA template. the thylakoid membrane of the chloroplast.
domain – A protein structure that may have separate sec- electronegativity – A concept derived from the relative
ondary structural elements that have a specific attraction of atoms across a bond. Atoms with
function (e.g., NADH binding). high values of electronegativity have a greater
double helix – The two strands of DNA polymers linked ability to attract bonding electrons.
together by hydrogen bonds. electrophoresis – A form of chromatography in which
double-reciprocal plot – see Lineweaver–Burk plot. the mobile phase is driven by an electric field
down regulation – A mechanism that decreases the num- imposed by electrodes; the stationary phase may
ber of receptors on a cell surface target, making be a gel composed of a polymer, such as poly-
the cell less sensitive to excess hormones or other acrylamide (see chromatography).
ligands by degrading or internalizing them. elongation – The process of extending a polymer, such as
downstream – Direction of a nucleic acid polymer that is an RNA chain or a protein.
further away from a given point (often the start emulsification – The dispersion of insoluble lipids (e.g.,
site) for transcription. triglycerides) into water solutions.
enantiomers – Molecules whose structures are non-
superimposable mirror images.
E endergonic – A positive change in free energy. An end-
ergonic reaction will only proceed in the reverse
EF hand – A 40-amino-acid-residue domain containing a direction to the one written.
helix-loop-helix design that binds Ca2+ ions with endoplasmic reticulum – A membrane network of tubules
high affinity. forming an enclosed space within the cell that is
eicosanoids – A group of endogenous lipid-signaling active in lipid transport, vesicular transport, pro-
molecules synthesized from arachidonic acid. tein export, and Ca2+ release to the cytosol.
electric field effect – A through-space effect within a endothermic – A change in enthalpy for a reaction that is
molecule due to electrically charged groups. positive (ΔH>0).
xxx Glossary
energy – A nonmaterial driving force for reactions, clas- diet. These amino acids are required for the for-
sified as potential (stored) or kinetic (expressed). mation of proteins.
Different forms of energy include electrical, grav- essential fatty acids – A group of unsaturated fatty acids
itational, heat, and mechanical. essential to human health that cannot be man-
energy coupling – The use of high energy molecules ufactured in the body, so they come from oils
within a reaction to ensure that it can proceed and fats found in plants and animals. Examples:
with a negative free energy. As a formal proce- omega-3 and omega-6 fatty acids.
dure, reactions that do not actually take place can evolution – The fundamental thesis of biology explain-
be summed using their standard free energies to ing how species arise: from previous species. The
arrive at an overall favorable free energy change. principle applies also to macromolecules, such
This formalism should not be confused with an as DNA and protein, which can be mapped from
actual mechanism, however. their earliest origins and indicate their relation-
enthalpy – A heat change for a system at constant pres- ship to other, similar forms.
sure; a defined energy change for thermodynamic excision repair – A process to correct DNA damaged
systems. The enthalpy change results largely from after replication by removing the faulty segment
bond energies in reactions. and, through DNA synthesis, replacing it with
enthalpy driven – A chemical reaction in which the free the correct segment replicated from a template of
energy change is dominated by the enthalpy term an undamaged DNA strand.
(ΔH). exciton transfer – A form of resonance energy transfer
entropy – An expenditure of energy that is unavailable that occurs between chlorophyll molecules in
to apply to work. The increased entropy is due to photosynthesis.
an increased dispersion of a system. For example, exergonic – A reaction for which ΔG < 0, where G is free
when two bonded atoms are split apart, the reac- energy. Every reaction in a functional metabolic
tion entropy increases. pathway is exergonic.
entropy driven – A chemical change in which the free exothermic – A change in enthalpy for a reaction that is
energy change is dominated by the entropy term negative (ΔH < 0).
(ΔS). exome sequencing – A laboratory technique for separat-
enzyme – A biological catalyst that lowers the activation ing the exons from the remainder of the DNA
energy for a reaction without affecting the equi- and then sequencing just those nucleotides.
librium position, enabling a reaction to proceed exons – A segment of a gene that contains information
with minimal changes in temperature. used in coding for protein synthesis. Genetic
enzyme activity – Measure of an enzyme function in information within genes is discontinuous, split
units of moles of substrate converted to product among the exons that encode for messenger RNA
per unit time. and absent from the DNA sequences in between
enzyme complex – A group of enzymes that cata- (see introns). Genetic splicing, catalyzed by
lyzes a metabolic sequence without releasing enzymes, results in the final version of mRNA,
intermediates. which contains only genetic information from
enzyme conservation – A statement that the total the exons.
amount of enzyme (Etot) is constant for the course exonuclease – An enzyme capable of detaching the ter-
of a reaction and can be expressed as the sum of minal nucleotide from a nucleic acid chain; any of
all enzyme forms. a group of enzymes that catalyze the hydrolysis
epimerase – A type of enzyme that catalyzes the stereo- of single nucleotides from the end of a DNA or
chemical rearrangement of hydroxyl groups in a RNA chain.
sugar. extremophiles – Organisms that thrive in extreme exter-
epimers – Sugars that differ at chiral centers other than nal environments, such as high temperature,
the D or L positions. pressure, or salinity.
epinephrine – A catecholamine hormone released by the
adrenal glands and some neurotransmitters of
the central nervous system in response to stress. F
equilibrium – A state that exists when the forward and
reverse rates of a reaction are equal. In chemis- fatty acids – Molecules with two parts: a long hydro-
try, it is when the products and reactants are in a carbon segment (the tail), and a smaller region
constant ratio. that typically consists of a carboxyl group (head).
ES – Intermediate enzyme–substrate complex. The hydrocarbon tail consists mostly of chemi-
essential amino acids – Any of the amino acids not syn- cally unreactive methylene groups with a variable
thesized by the body and so must come from the number of double bonds.
Glossary xxxi
Faraday – An electrical conversion constant converting amino acids that make up each protein synthe-
numbers of electrons to mols. sized in the cell.
feed-forward activation – A modulator (stimulation or glucagon – Hormone secreted by the α-cells of the pan-
inhibition) of an enzyme that arises from a reac- creas during fasting conditions, as blood glucose
tion that occurs earlier in the same metabolic concentrations are decreased. Glucagon acts
pathway. on the liver to increase glucose release into the
fermentation – An early name for yeast glycolysis, which bloodstream.
produces ethanol and CO2. glucogenic – Giving rise to or producing glucose.
ferredoxin – A mobile protein electron carrier in pho- glucokinase – Isozyme of hexokinase with a very high Km
tosynthesis that slides along the stromal surface (20 mM) for glucose.
of the thylakoid membrane of the chloroplast, gluconeogenesis – A process by which glucose is made in
accepting electrons from PSI, and donating to the liver from noncarbohydrate precursors (e.g.,
NADP+ (linear photosynthesis) or cyt. b6f (cyclic lactate, amino acids, and glycerol).
photosynthesis). glucose-6-phosphatase – An enzyme that catalyzes the
Fischer projection – A two-dimensional representation hydrolytic dephosphorylation of glucose-6-phos-
of the three-dimensional structure of an organic phate, which is the principal route for hepatic
molecule. gluconeogenesis, thus allowing glucose stored in
5' end – One end of a linear polynucleotide strand at which the liver to enter the blood.
the 5'-hydroxyl group of the terminal nucleoside glutathione – A peptide composed of glycine, cysteine,
residue is not joined to another nucleotide (i.e., and glutamic acid. Reduced glutathione (due to
the free end). the cysteinyl –SH group) reacts with intracellu-
flavonoids – A large group of water-soluble plant pig- lar proteins in order to maintain their sulfhydryl
ments that are polyphenols that have reputed groups in the reduced form, part of the antioxi-
antiviral, antioxidant, and anti-inflammatory dant system in cells.
health benefits. glycerol phosphate shuttle – A mechanism for the
fluid phases – Forms of matter that assume the shape transport of electrons from cytosolic NADH to
of their container. These include both gases and mitochondrial carriers of the oxidative phos-
liquids. phorylation pathway.
folic acid – A water-soluble vitamin (classified as a B glyceroneogenesis – Metabolic pathway occurring in
vitamin) that is a precursor to tetrahydrofolate adipose tissues and liver in mammals that can
(THF) and dihydrofolate (DHF), cofactors in convert pyruvate to the glycerol of triacylglycerol
one-carbon metabolism. (the major storage form of fat).
fractional saturation – Proportion of binding of a ligand glycogen – A branched polysaccharide that is the
to a macromolecule, such as oxygen binding to major form of carbohydrate storage in animal
myoglobin or hemoglobin. cells.
free energy – A thermodynamic term that describes the glycogen branching enzyme – In glycogen synthesis,
overall directional change of a reaction, combin- the enzyme that catalyzes the transfer of a lin-
ing the first law (energy changes as enthalpy) and ear (αl→4) segment of the molecule to a hydroxyl
the second law (entropy). group at the 6 position to create a branch.
fructose bisphosphatase – Hydrolase enzyme converting glycogen phosphorylase – In glycogenolysis, the major
the bisphosphate of fructose into fructose-6-P. enzyme leading to a release of glucose-1-phos-
phate from glycogen.
glycogen phosphorylase kinase – An enzyme that cata-
G lyzes the conversion of phosphorylase b to phos-
phorylase a (the phosphorylated, active form);
galactose – A sugar that is the 4-epimer of glucose. activated by Ca2+ and cyclic AMP dependent
galactose catabolism – Breakdown of galactose, converg- protein kinase.
ing with the glycolytic pathway at glucose-6-P. glycogen storage diseases – A set of inborn genetic
gas liquid chromatography – An analytical technique errors of metabolism deleterious to glycogen
where the sample and carrier fluid are con- metabolism.
verted into the gas phase (the mobile phase); glycogen synthase – An enzyme that catalyzes the trans-
the stationary phase is packed into a long, thin fer of a glucosyl unit from uridine diphosphate
column. glucose (UDP-glucose) to glycogen.
genetic code – The rules for matching of codons to their glycogen synthase kinase (GSK) – A cytosolic pro-
amino acids. In the polynucleotide chain, the tein kinase that catalyzes the phosphorylation
pattern of nucleotides governing the sequence of
xxxii Glossary
of serine residues in glycogen synthase and cytochromes, or at fixed oxidation state, as in

inactivates it. hemoglobin.
glycogen synthesis – Pathway of formation of glycogen heme regulated inhibitor – A protein kinase that sup-
from individual glucosyl subunits. presses protein translation in reticulocytes when
glycogenin – A protein at the core of the glycogen par- heme or iron concentration is low.
ticle, linked to the glucose subunit through a hemiacetals – The condensation product of an alco-
tyrosine residue. hol and an aldehyde. The hydroxyl group of
glycogenolysis – Degradative pathway converting glyco- the hemiacetal is used to form glycosides in
gen to glucosyl subunits (glucose-1-P). sugars.
glycosidic bond – The molecular link between sugars, hemiketals – The condensation product of an alcohol and
chemically an acetal or a ketal. a ketone. The hydroxyl group of the hemiketal is
gout – A metabolic arthritis resulting from over-produc- used to form glycosides in sugars.
tion of uric acid (see allopurinol). hemimethylated – A DNA chain that has a methyl group
G protein – Signaling proteins that are either bound to on one strand but not the other.
GTP (active form) or GDP (inactive form). On the high-energy electrons – The electrons carried in redox
inner surface of plasma membranes, trimeric G transfer cofactors such as NADH that ultimately
proteins transmit some hormone signals to their lead to energy generation.
effectors such as in the production of cyclic AMP. high-energy molecules – Molecules containing local-
Within cells, small G proteins act as intermedi- ized electrons that can be redistributed and used
ate control systems, such as those regulating pro- as an energy source in a further reaction, such as
tein synthesis. NADH and ATP.
grana – Overlapping stacks of membranes within the high-energy phosphate – A phosphoryl group that is
chloroplast organelle. exchanged between molecules, usually involving
at some point the mobile cofactor intermediate,
ATP.
H high pressure liquid chromatography (HPLC) – An
analytical technique using many small beads and
hairpin turn – In RNA transcripts that have a palin- a correspondingly large surface area for the sta-
dromic sequence, the formation of a double- tionary phase. High pressure is needed to drive
stranded segment functions as a control signal flow of the mobile phase (see chromatography).
for protein-nucleic acid interactions. histones – Small basic proteins that bind the negatively
half-cell – One of two solutions of electrolytes with an charged backbone of DNA and contribute to the
electrode enabling separate oxidation and reduc- condensation and packing of DNA.
tion in a galvanic cell. holistic – A view that focuses on system properties rather
half-reaction – The oxidation or reduction portion of a than its parts.
redox reaction, either a formal representation, or hydrated – Attached to water molecules.
a physical one within a half-cell. hydride ion – The anion of hydrogen (H:-).
Haldane – An equilibrium constant for an enzyme hydrogen bond – An interaction involving a hydrogen
expressed in terms of kinetic constants rather atom attached to a N or O atom and another
than rate constants. N or O atom. The bond strength is intermedi-
Haworth projection – A perspective representation of ate between van der Waals forces and covalent
the molecular ring structure of sugars, using pen- bonds.
tagons, hexagons, and shading to depict bonds in hydrolases – A category of enzymes that are actually a
three dimensions. type of transferase (itself a separate category of
heat – A form of energy that transfers among molecular enzymes) in which water is a substrate.
particles through the kinetic force of those parti- hydrophobic effect – A clustering of nonpolar mol-
cles, always in the direction of high temperature ecules driven by more favorable water–water
to low temperature. interactions.
helix breakers – Amino acids that disturb the ability of
a protein segment to form an alpha helix, either
through restricted bonding (like proline), too I
much rotational freedom (glycine), or neighbor-
ing charge interactions (like multiple proximal immunophilins – Drugs that form a complex with prolyl
glutamate residues). isomerase and then inhibit specific targets, such
heme – An iron binding cofactor made up of linked as calcineurin, a protein phosphatase essential to
imidazoles. The iron may be redox active, as in T-cell proliferation in the immune system.
Glossary xxxiii
inhibitor – A substance that decreases the rate of an produced by covalent incorporation of the mol-
enzymatic reaction. ecule into the enzyme.
insulin – A hormone secreted from the cells of the isolated double bond – A type of double bond in which
pancreas into the bloodstream in response to the π orbitals do not overlap with those of other
an increase in blood glucose. Insulin leads to a double bonds (alternatively, nonconjugated).
decrease in blood glucose concentrations by isoelectric point – The pH at which a molecule has a net
stimulating glucose uptake and metabolism in zero charge.
cells. isoforms – Distinct proteins that function identically;
insulin receptor substrate 1 (IRS-1) – A cytosolic regu- isomeric form of the same protein, with different
latory protein that is phosphorylated by the insu- amino acid sequences but with the same activity.
lin receptor and serves as a binding site for other isomerases – A class of enzymes that catalyze rearrange-
regulators in the cell. ment reactions, such as the interconversion of
insulin-resistant – A condition in which cells respond ketoses and aldoses.
poorly to the presence of insulin, found almost isozymes – Enzymes with different structures that cata-
universally in obese individuals, and a precursor lyze the same reaction.
state to diabetes.
initial velocity – The first (linear) portion of a reaction
progress curve, either disappearance of substrate, K
or appearance of product. In theory, the slope of
this curve as time approaches zero is defined as ketogenic – Amino acids that are catabolized to acetyl-
the initial velocity; in practice, the first 5% to 10% CoA and thus potentially to ketone bodies.
of the progress curve is approximately a straight ketone bodies – Products of partial lipid oxidation
line. between liver and target tissues such as brain,
inner mitochondrial membrane – One of two mem- heart, and skeletal muscle. Acetoacetate and
branes in the mitochondrion, this membrane is β-hydroxybutyrate are the major ketone bodies.
infolded, providing a greater membrane area for ketoses – Sugars in which the carbonyl group is a ketone,
the components of oxidative phosphorylation located at carbon two.
and transport proteins (see outer mitochondrial kinetic constant – A collection of rate constants that
membrane, matrix space). characterizes enzymatic rate equations.
inorganic phosphate – An isolated phosphate group that Km – A kinetic constant that is a substrate concentration
is a weak acid and can combine with an alcohol occurring at half of the maximum velocity of an
to form an ester. enzyme.
intermediary metabolism – Connected reaction Krebs cycle – One of the major metabolic pathways in
sequences in cells that represent the major cellular metabolism. A cyclic pathway that con-
pathways, common to cells in many biological verts acetyl CoA to CO2 and extracts energy
organisms. intermediate molecules NADH, UQH2, and GTP
internal energy – The energy of the system under study, (see citric acid cycle, tricarboxylic acid cycle).
combining the amount of heat put into a system
with the amount of work delivered by the system.
intrinsic factor – A protein found in cells of the stomach L
lining (epithelia), secreted into the stomach, and
used later in the small intestine in the uptake of lac operon – A grouping of genes and regulatory prod-
dietary vitamin B12. ucts in bacteria that controls the synthesis of
introns – A segment of a gene situated between exons three enzymes that enable utilization of lactose
that does not function in coding for protein syn- as a carbon source.
thesis. After transcription of a gene to messenger lactose intolerance – A common genetic deficiency of
RNA, the introns are removed, and the exons are lactase (an enzyme that catalyzes conversion of
spliced together by enzymes before translation lactose to galactose and glucose) that impairs the
and assembly of amino acids into proteins (see digestion of dairy products. The partial bacte-
exons). rial oxidation of lactose in the large intestine is
ionization of water – The dissociation of water into responsible for the symptoms of bloating and gas
hydrogen ions (H+) and hydroxide ions (OH−). production.
ionophore – A molecule that binds an ion and transports laforin – A phosphatase active on glycogen polymer,
it across a membrane. defective in Lafora disease.
irreversible inhibition – Inhibition that is not charac- Lafora disease – A rare disorder of glycogen metabo-
terized by an equilibrium constant, but is instead lism in which large and hyper-phosphorylated
xxxiv Glossary
glycogen particles appear in brain and other between the faces of the mitochondrial mem-
tissues. brane (see Q cycle).
lagging strand – In DNA replication, the strand in which lovastatin – Inhibitor of hydroxymethylglutaryl CoA
the nascent strand is synthesized in discontinu- reductase and thus cholesterol synthesis; it is a
ous segments because all transcription must be natural product isolated from a fungus used to
in the 5' to 3' direction and both DNA strands treat hypercholesterolemia.
(oppositely arranged) must be replicated (see lumen – Most interior water space of the chloroplast,
Okazaki fragments, leading strand). bounded by the thylakoid membrane. Also, a
leading strand – In DNA replication, the strand that is general anatomical term for an extracellular
synthesized continuously during replication (see space interior to the body, such as the lumen of
lagging strand). the intestines.
Le Chatlier principle – A postulate that reactions lyase – A category of enzymes that catalyze the splitting
proceed in the direction of greater to lesser of a substrate into pieces; lyase reactions do not
concentrations, compared with equilibrium involve water as a substrate.
concentrations.
lecithin – A common name for phosphatidylcholine.
leghemoglobin – A monomeric heme containing pro- M
tein structurally similar to myoglobin, found in
the root nodules of leguminous plants (such as macromolecules – A large molecular polymer such as a
soybeans) that are host to nitrogen-fixing bacte- protein that exhibits new properties that distin-
ria. Oxygen inhibits nitrogenase; leghemoglobin guish it from small molecules.
sequesters oxygen, keeping its concentration low, major groove – One of two grooves along the DNA
but still able to supply bacterial respiration. double helix, this has the larger gap between the
Lewis acid or base – Acids defined as electron pair accep- sugar base structures when viewed from the top
tors; bases defined as electron pair donors. of the helix structure (see minor groove, double
leukotrienes – A group of biologically active compounds helix).
derived from arachidonic acid that are respon- malate/aspartate shuttle – A pathway for importing
sible for allergic and inflammatory reactions. NADH from the cytosol to the mitochondria,
light reactions – Steps in photosynthesis in which light using reduction of oxalacetate to malate in the
energy is utilized to produce the high energy cytosol, malate to oxalacetate in the mitochon-
intermediate compounds ATP and NADPH. drial matrix, and interconversion between
lipid droplet – An intracellular organelle in which the oxalacetate and aspartate; neither NADH
interior is largely triacylglycerols and other nor oxalacetate can cross the mitochondrial
lipid molecules, with a monolayer phospholipid membrane.
membrane. malin – A part of the ubiquitin system (a ligase attaching
Lineweaver–Burk plot – Double reciprocal transform the protein ubiquitin) defective in Lafora disease.
plot of the Michaelis–Menten equation, which maple syrup urine disease – A genetic disease caused
linearizes the resulting graph (also termed dou- by the accumulation of three amino acids – leu-
ble-reciprocal plot ). cine, isoleucine, and valine – due to their inabil-
lipids – Relatively unreactive hydrocarbons such as oils ity to be oxidized through the branched chain
and fats; they are entirely or mostly water insolu- ketoacid dehydrogenase complex. Named for the
ble. Functions include energy storage and mem- distinct burned sugar odor of urine (also termed
brane formation. branched chain ketoaciduria ).
liposomes – A spherical particle formed by a lipid bilayer mass action ratio – The ratio of the reaction products
enclosing an aqueous compartment; used to con- multiplied together to the substrates multiplied
vey viruses, drugs, and other substances. together for a reaction at any given point. When
loop – Describes the shape of a type of secondary struc- the forward and reverse reaction rates are the
ture of protein formed with the backbone atoms same, the mass action ratio is equal to the equi-
and their hydrogen bonds that link other sec- librium constant.
ondary structures together (also termed random mass spectrometry – A system for identifying molecules
coil). by fragmenting them into ions in the gas phase
loop mechanism – A mechanism for creating the proton and then separating them in an electric field by
gradient in the Q cycle, involving the movement mass.
of oxidized and reduced ubiquinone moving matrix space – The water space enclosed by the mito-
chondrial inner membrane.
Glossary xxxv
melanosomes – Lysosome organelles that contain the enzyme and the enzyme-substrate complex (also
pigment melanin. called noncompetitive inhibition).
melting curve – A plot of strand separation of DNA with mobile cofactors – Part of an enzymatic reaction that
increasing temperature. can dissociate from the enzyme at the conclu-
melting temperature – The midpoint of the DNA melt- sion of the reaction and participate in a reaction
ing curve. in a separate pathway in the cell. Examples are
membrane fluidity – The relative ability of individual NADH, UQH2, and cytochrome c. By contrast,
molecules to move within the bilayer of a cell. bound cofactors never leave the enzyme during
mesophyll cells – Cells of C4 plants found in an outer the catalytic cycles.
ring that capture CO2 for RuBisCo by carbox- molecular biology – A broad term encompassing genet-
ylation of PEP, and transporting subsequently ics, biochemistry, and growth processes of the
formed malate into the inner ring cells (bundle cell.
sheath) which is where RuBisCo is located. monomers – Individual protein chains (subunits).
metabolically irreversible – Metabolic reactions within motif – Here used as a synonym for domain. Some inves-
cells that are greatly displaced from their equi- tigators consider a motif as a specific portion of
librium positions and never run in the reverse a domain.
direction under cellular conditions. These reac- mTOR – A protein kinase (positively) regulating pro-
tions are the sites of metabolic control, through tein synthesis by catalyzing phosphorylation of
allosteric or covalent modification. control proteins such as S6K. Originally named
methionine cycle – Metabolic pathway of one carbon as “mammalian target of rapamycin”, it is also
metabolism in which methionine is converted named “mechanistic target of rapamycin”.
to S-adenosyl methionine and a methyl group is muscle hypertrophy – Increase in the size of muscle
donated to an acceptor. tissue.
methylcelluloses – A biologically inert polymer formed
in the laboratory by creating methyl esters to
some of the hydroxyl groups of cellulose; their N
partial solubility in water can be controlled by
the extent of methylation. N-acetylglutamate – A compound produced from acetyl
micelles – A form taken by lipid amphiphiles in water coenzyme A and glutamate; an allosteric acti-
solution in which the polar portions of the mol- vator of carbamoyl phosphate synthetase in the
ecules face the aqueous exterior and the nonpo- urea cycle.
lar portion forms an interior core excluded from NAD binding domain – The site of binding of this mobile
water. cofactor, common to a large number of enzymes.
midpoint potential – The redox potential for a reaction near-equilibrium – A reaction in cells that has a mass
under (biological) standard conditions. action ratio close to the equilibrium constant,
minor groove – One of two spacings along the DNA dou- and can run in the reverse direction in a separate
ble helix, this has the smaller gap between the pathway. Such reactions are controlled by sub-
sugar base structures when viewed from the top strate and product concentrations and are not
of the helix structure (see major groove, double regulated by external means (i.e., by allosterism
helix). or covalent modification).
mismatch repair – A system within the cell during rep- negative cooperativity – Cooperativity in which suc-
lication that corrects errors in DNA by detecting cessive ligand molecules appear to bind with
and replacing bases in the DNA that are wrongly decreasing affinity.
paired; an enzyme catalyzes removal of the mis- negative supercoil – Occurs when the double helix
matched segments, and then DNA polymerases strand of DNA is twisted in a counterclockwise
fills the gaps in the strands with the appropriate direction (the opposite direction to turns in the
bases. helix), which either unwinds or twists the entire
mitochondria – Organelle evolutionarily derived from structure (see supercoils, double helix).
bacteria, responsible for most of the energy gen- Nernst equation – Expression of the reaction potential
eration of the cell through oxidative phosphory- derived from the free energy equation; this equa-
lation. Mitochondria contain portions of several tion relates the redox potential to the reaction
metabolic pathways, such as urea synthesis and concentrations.
gluconeogenesis. nitrification – A process where the oxidation of ammo-
mixed inhibition – A type of reversible enzyme inhibi- nia (NH3) to nitrites and nitrates is accomplished
tion in which the inhibitor binds both the free by bacteria in a series of reactions.
xxxvi Glossary
nitrogen cycle – A global nitrogen cycle by which atmo- operon – A group of genes or a segment of DNA that
spheric nitrogen gas (N2) is fixed into nitrogen functions as a single transcription unit, com-
oxides and ammonia, which can be incorporated prised of an operator, promoter, and one or more
into biological systems, and then converted back structural genes that are transcribed into one
to N2. polycistronic mRNA.
nitrogen fixation – The incorporation of atmospheric osmosis – The ability of water to move across a semi-
nitrogen, catalyzed by nitrogenase into ammonia permeable membrane due to differences in solute
by various bacteria; the process whereby certain concentrations across the membrane.
microorganisms, such as rhizobia, convert atmo- osmotic pressure – The applied pressure needed to
spheric nitrogen into compounds that plants and equalize the fluid levels across a semipermeable
other organisms can assimilate. membrane that exhibits osmotic imbalance.
nitrogenase – An enzyme complex converting N2 to outer mitochondrial membrane – One of two mem-
NH3. branes in the mitochondrion; porous to mole-
non-cooperative – A binding process in which ligands cules with a molecular weight up to about 10,000
bind independently to monomers of a multimeric (e.g., ions, ATP, ADP, and nutrient molecules)
protein. (see inner mitochondrial membrane).
nonessential amino acids – Amino acids that can be oxidation number – An assignment to an atom indicat-
synthesized by the organism and, thus, do not ing what its charge would be if it were not engaged
need to be present in the diet (see essential in a covalent bond. Useful in determining redox
amino acids). states of organic molecules.
nonpolar lipids – Lipids that are not amphiphiles. oxidative phosphorylation – The oxidation of reduced
Example: triacylglycerols. substances tied to the production of ATP by
nonreducing end – The sugar residue at the end of a poly- mitochondria.
saccharide that has no free anomeric hydroxyl oxidoreductases – A category of enzymes in which the
group. substrate changes the oxidation state during
nonreducing sugars – Any saccharide that has no free the process of going to product; there must be a
anomeric carbon atoms. Examples: sucrose and mobile cofactor that removes or adds those elec-
trehalose. trons, often NAD+.
N side – Negatively charged side of an energy producing
vesicle (chloroplast, bacteria, or mitochondria)
produced when hydrogen ions leave this space. P
nuclear magnetic resonance (NMR) spectroscopy –
A method for the study of molecular structure P – Phosphoryl group, often referred to as a phosphate
in which a magnetic field is imposed and the group, such as in the carbohydrate derivative glu-
absorption of electromagnetic radiation at spe- cose-6-phosphate (glucose-6-P).
cific frequencies is measured. palindrome – A self-complementary nucleic acid
nuclear membrane – The double-layered membrane sur- sequence. In general, any sequence of letters that
rounding the nucleus of a cell. reads the same in the forward direction as in the
nucleotides – Modified sugar molecules that contain a reverse.
nitrogenous base and at least one phosphate ester; parallel – In a β-sheet of a protein, when the strands run
the most common are AMP, ADP, and ATP, key in the same direction.
energy transfer molecules widely used in meta- PARP – see poly ADP-ribose polymerase.
bolic reactions in cells. Additionally, nucleotides partially ionic bond – A description of a covalent bond
form the polymers DNA and RNA. that emphasizes its partial ionic character due
to electronegativity differences of the atoms on
either side of the bond.
O Pasteur effect – A 19th-century observation by Louis
Pasteur that glucose utilization is decreased in
oil – The liquid form of lipids. the presence of oxygen in yeast metabolism.
Okazaki fragments – During DNA replication, the rela- path-dependent – A change in a system that varies
tively short fragments of DNA synthesized on depending on how it is accomplished. For exam-
the lagging strand; later these fragments will be ple, the amount of work accomplished in a pro-
covalently connected into a continuous strand. cess depends on the route taken.
oligosaccharides – A carbohydrate whose molecules are path-independent – A change from one state to the next
composed of a relatively small number of mono- that is independent of the route that is taken. This
saccharides linked via glycosidic bonds. describes several changes in thermodynamics
Glossary xxxvii
such as the change in internal energy, enthalpy, phosphonate – Phosphate in which one of the P–O bonds
and free energy. is substituted by a P–C bond.
pathway completion – In a metabolic pathway, all mobile phosphoribosylpyrophosphate (PRPP) – This multiply
cofactors must be balanced so that the route can phosphorylated ribose is a precursor in the syn-
be traversed again by further pathway substrates. thesis of nucleotides.
pathway flux – The overall rate of a pathway that is also phosphotyrosine binding domain – Domains of 100–
the rate of every individual reaction in the path- 150 residue modules that commonly bind the
way under a given steady-state condition (see Asn-Pro-X-Tyr motifs where the tyrosine residue
pathway substrate, pathway product). is phosphorylated. Found in proteins involved in
pathway-irreversible – A modification of an enzyme regulatory cascades.
that cannot be reverted by a separate reaction(s) photosynthesis – Pathway for utilization of light energy
in a cell, and is thus a permanent change until the to form high energy intermediates (the light reac-
enzyme is degraded by proteolysis. tions); usually also refers to the ultimate synthe-
pathway product – The final product of a series of reac- sis of carbon compounds such as carbohydrates
tions that defines a pathway in cells. (see carbon reactions).
pathway substrate – The initial product of a series of photosynthetic – Type of organism that can utilize light
reactions that defines a pathway in cells. to synthesize carbon compounds.
pathway-reversible – A modification of an enzyme that photosystem (PS) – Complex of proteins involved in pho-
can be reverted by a separate cellular reaction tosynthetic energy transfer that accept light input
without requiring proteolysis. The most common and boost electron energy of other molecules.
example is protein phosphorylation, reverted by a photosystem I (PS I) – In green plant photosynthesis,
protein phosphatase. contains iron-sulfur proteins and transfers elec-
PCR – see polymerase chain reaction. trons from plastoquinone to ferredoxin.
pentose phosphate shunt – Pathway branching from photosystem II (PS II) – In green plant photosynthesis,
glycolysis at glucose-6-P that produces ribose contains an iron center and transfers electrons
carbon for nucleotides and NADPH. from water to plastoquinone.
peptides – Short chains (roughly 25) of amino acids that photon – Minimal unit of light energy defined from the
have sufficient flexibility in solution so that a equation E = hν where E is energy, h is the Planck
fixed higher order structure does not exist. constant, and ν is the frequency of light.
peptide bond – An amide linkage found between the phylloquinone – A form of Vitamin K.
amino acids of proteins. ping-pong (mechanism) – An enzyme mechanism in
phenylketonuria – A genetic disorder in humans caused which one substrate binds and the product is
by an inability of the body to metabolize phenyl- released before a second substrate binds. This
alanine to tyrosine. mechanism occurs in transaminase reactions.
pheophytins – Part of the reaction center in the light PKC – see protein kinase C.
reactions of photosynthesis, these molecules are PKU – see phenylketonuria.
structurally similar to chlorophyll molecules plasmodesmata – A bridge of cytoplasm that spans adja-
except that the chelate ring has no metal inside it cent plant cells to permit communication and
( see reaction center). transport between individual cells. Gap junctions
phosphatidic acid – A phospholipid with a single fatty are the functional equivalents in animal cells.
acyl chain attached to a glycerol phosphate polar bond – A bond between two atoms that have large
backbone. differences in electronegativity.
phosphatidylinositol phosphate 3-kinase – An enzyme polarimeter – A device that detects chiral molecules and
that catalyzes the incorporation of a phosphoryl differentiates the two forms by determining the
group into the 3-position of the inositol ring. rotation of the plane of polarized light as it passes
phosphodiesterase – An enzyme that cleaves phos- through a solution.
phodiesters into a free hydroxyl group and a polar lipids – Lipid molecules that are amphipathic, with
phosphomonoester. one portion of the molecule interacting with
phosphoenolpyruvate carboxykinase (PEPCK) – The water. These include cell membrane lipids.
rate-limiting step of gluconeogenesis under most polar molecule – Describes a molecule with a net polar-
conditions, this enzyme converts oxaloacetate to ity due to the overall vector sum of its bonds.
carbon dioxide and phosphoenolpyruvate. polyamines – Molecules with multiple amines, bearing
phospholipid-dependent kinase – A regulatory enzyme a net positive charge and interacting with DNA
that phosphorylates serine and threonine resi- through electrostatic attraction. The polyamines
dues in response to protein kinase B and mem- are: putresciene, spermidine, and spermine.
brane phospholipids.
xxxviii Glossary
poly-A tail – A structure of up to 250 adenylate residues primitive – A concept that has no deeper understand-
located on the 3' end of eukaryotic mRNAs used ing and is accepted on faith as there have been
in translation and protecting the mRNA against no contradictions. For example, the concept of
nuclease action. charge is a primitive, as we understand its quali-
poly A polymerase – An enzyme catalyzing the incorpo- ties but nothing further.
ration of a string of adenosine residue on the 3’ primase – An RNA polymerase that catalyzes comple-
end of mRNA. mentary ribonucleotide base additions to single-
poly ADP-ribose polymerase – An enzyme catalyzing stranded DNA in the first biochemical reactions
the incorporation of multiple copies of the ADP- to add bases to the strand.
ribose portion of NAD+ into proteins involved in primosome – During DNA replication, the protein
DNA repair. The poly-ADP tail is a binding site complex containing primase and helicase that
for other repair proteins. initiates the RNA primers on the lagging DNA
polycistronic – Bacterial mRNA that contains sequences strand.
for several different proteins in the same messen- probenecid – A drug used to treat gout by increasing
ger RNA strand. urate excretion by the kidneys (see gout).
polymerase chain reaction – A technique for amplify- processivity – The movement and catalysis of a mol-
ing small quantities of DNA by multiple rounds ecule on a substrate surface without dissociation
of synthesis of complementary chains, heating to between catalytic cycles.
release the finished chains, and cooling to syn- prochiral – An achiral molecule that can be enzymati-
thesize chains. The polymerase enzyme is iso- cally converted to a chiral one. Prochiral mol-
lated from a thermophile (an extremophile). ecules all have double bonds at positions that
polymers – Large molecules composed of many repeat- become chiral centers.
ing subunits. promoters – A site in a DNA molecule at which RNA
polyprotic acid – An acid that can donate more than one polymerase and transcription factors bind to ini-
proton, such as phosphoric acid. tiate transcription of messenger RNA.
polysaccharides – A carbohydrate polymer composed prosthetic group – A small molecule that is bound to
of monosaccharide residues bound together via a protein to assist its function. These molecules
glycosidic bonds. remain bound to the protein surface as it func-
polyunsaturated – Fatty acids with more than one dou- tions, and may be considered an extension of the
ble bond. protein itself.
porins – Protein molecules lining the outer membrane of protease – An enzyme that hydrolyzes peptide bonds in
the mitochondria, which enable passage of mol- proteins.
ecules up to 10,000 Da. (see also VDAC) proteasome – A cytosolic protein complex in which pro-
positive cooperativity – Cooperativity in which suc- teins enter and are degraded to small peptides.
cessive ligand molecules appear to bind with protein – Linear chains of amino acids that form a spe-
increasing affinity. cific three-dimensional structure (thus distin-
positive supercoil – Occurs when the double helix strand guishing them from smaller peptides that are
of DNA is twisted in a clockwise direction (the usually on the order of 25 amino acids or less).
same direction as the turns in the helix), which Proteins are the most diverse biomolecules,
tightly coils the structure (see supercoils, double including virtually all of the enzymes, the bio-
helix). logical catalysts.
postprandial state – A period immediately after the protein kinase C – A protein kinase that catalyzes the
ingestion of a meal. phosphorylation of serine or threonine amino
post-translational modification – A covalent modi- acid residues. The “C” in the name derives from
fication to a protein apart from its ribosomal an early finding that a calcium-stimulated prote-
synthesis. ase activated the enzyme; however, this proteoly-
prenylation – The incorporation of branched chain lip- sis is not the mechanism for its rapid activation
ids (isoprene derivatives) into proteins that com- in cells. An increase in the lipid mediator diacyl-
monly enables interaction with membranes. glycerol is the activator of PKC.
Pribnow box – A highly conserved sequence element proton motive force – The driving force for oxidative
located upstream from the start site of transcrip- phosphorylation, composed of a chemical (pH)
tion, to which the sigma subunit of RNA poly- gradient and an electrical gradient, both due to
merase binds; found in prokaryotes, such as E. a difference of hydrogen ions across the inner
coli and bacteriophage genes. mitochondrial membrane.
primary structure – The sequence of amino acids in a PS I – Photosystem I of chloroplasts (see photosystem).
linear protein chain. PS II – Photosystem II of chloroplasts (see photosystem).
Glossary xxxix
P side – Positively charged side of an energy producing rate constant (k) – The proportionality between velocity
vesicle (chloroplast, bacteria, or mitochondria) and substrate concentration for a reaction.
established when hydrogen ions enter this space. reaction center – In photosynthesis, a special pair of
purine nucleotide cycle – A metabolic cycle between chlorophyll molecules that accept energy trans-
AMP and IMP that serves to convert aspartate ferred from other chlorophyll molecules.
into fumarate as an anapleurotic (refilling) reac- reactive oxygen species – Molecules formed ultimately
tion for the Krebs cycle. from oxygen (a diradical) that contain unpaired
pyridoxal phosphate – A cofactor derived from vitamin electrons and can cause cellular damage.
B6 that is the prosthetic group of glycogen phos- receptor tyrosine kinases – A class of membrane recep-
phorylase and amino acid transaminases. tors that have intrinsic kinase activity selective
pyrrole – A five-member heterocycle containing one for tyrosine residues.
nitrogen atom and four carbon atoms. redox – Oxidation-reduction reactions involving an elec-
pyruvate carboxylase – An enzyme that catalyzes the tron transfer.
carboxylation of pyruvate to oxalacetate, impor- reducing end – The sugar residue at the end of a poly-
tant in lactate gluconeogenesis, fatty acid synthe- saccharide that has an equilibrium form with an
sis, and as an anapleurotic reaction for the Krebs open-chain aldose or ketose.
cycle. reducing equivalents – Energy-rich electrons that can
pyruvate dehydrogenase complex – A noncovalent be transferred from cofactors and utilized for
association of enzymes that converts pyruvate to energy extraction or reduction of compounds,
acetyl CoA. such as in the hydride NADH.
pyruvate transporter – In the inner mitochondrial reducing sugar – Any saccharide that has at least one free
membrane, transports pyruvate into the mito- anomeric carbon (i.e., an anomeric carbon not
chondrial matrix space. involved in a glycosidic bond). A small amount
of this open-chain form exists in solution, so its
carbonyl group can reduce certain metal ions in
Q diagnostic tests.
reductases – Enzymes that catalyze the reduction of their
Q cycle – The mechanism for complex III production substrate.
of the proton gradient across mitochondria in reductionist – A “bottom up” perspective; in biochemi-
which oxidized and reduced forms of ubiquone cal terms, focusing on specific reactions or
(UQ, UQH2) move between the membrane faces chemical species or decomposing a more com-
and ferry protons across the membrane. plex system into its elements in order to study
quaternary structure – A structural level for proteins finer detail.
that have more than one protein chain. reduction potential – A measure of the capacity of a
redox half-reaction to donate electrons.
repetition-variation – A process that is repeated with a
R slight variant, as the Krebs cycle and other meta-
bolic reactions.
racemase – An enzyme that catalyzes the inversion replication – The process by which DNA can be used as
around an asymmetric carbon atom. a template to form daughter DNA molecules for
racemic mixture – A mixture of two optical isomers in cell division.
solution that shows no net optical activity. replication fork – The section of a DNA molecule at
radical anion – A molecule containing both a free radical which the two strands are just separated; here,
and a negatively charged ion. both daughter molecules are being synthesized.
raffinose – A sugar found in foods such as cabbage and replisome – In DNA replication, a protein complex
beans that is poorly digested due to the presence containing two DNA polymerase molecules for
of the galactose (α1→ 6) glucose bond in the first synthesizing the leading and lagging ends of the
two sugar moieties of the trisaccharide; its bacte- DNA strands.
rial degradation in the large intestine causes gas. resistance training – Exercise that leads to increased
random coil – A protein segment with no recognizable protein synthesis in muscle, involving fast (gly-
structure of its own linking secondary structures colytic) muscle.
in proteins. restriction enzyme – Bacterial DNA hydrolysis enzymes
ras oncogene – A G-protein gene having an inactive derived from a bacterial defense from bacterio-
GTPase, which leads to a state of constant activa- phage. The enzymes recognize specific sequences
tion and hyperactive cell growth, and is a cause in DNA and are invaluable for virtually all phases
of cancer. of molecular biology.
xl Glossary
rho protein factor – A protein involved in prokaryotic sequential mechanism – An enzyme mechanism with
transcription termination, which binds and dis- multiple substrates in which all must bind
assembles the RNA transcript with concomitant to the enzyme in turn before any product is
ATPase activity. released.
resonance – In conjugated double bonds, where electrons SH2 – see src homology 2.
can spread out over more than two nuclei. Shine–Dalgarno sequence – Located on a prokaryotic
respiration – Energy linked process in cells that utilizes mRNA molecule upstream of the translational
oxygen to enable electron flow that ultimately start site, the short (3–10), purine-rich sequence
leads to the formation of ATP. of nucleotides that binds ribosomal RNA, thereby
rhodopsin – Molecule formed from the covalent link- aligning the ribosome on the initiation codon on
ing of retinal with the protein opsin, respon- the messenger RNA. Proposed by John Shine and
sible for light absorption in the visual cycle of Lynn Dalgarno in 1975.
animals. shunt pathway – A pathway that branches from another
ribosome – A cytosolic complex of proteins and pathway and terminates at some other point in
ribosomal RNA (rRNA) that binds mRNA that pathway. Example: the 2,3-bisphosphoglyc-
and tRNA and functions in the synthesis of erate shunt of glycolysis.
proteins. shuttles – A pathway for transferring portions of a com-
ribozymes – An RNA segment able to catalyze the cleav- pound across membranes. Examples: the malate/
age and formation of covalent bonds in specific aspartate shuttle for reducing equivalents; the
sites of RNA strands; ribosomes are a nonprotein citrate shuttle for acetyl-CoA.
type of enzyme. signal sequence – During protein translation, several
rotor – The moving (spinning) part of a motor mecha- amino acid residues at the amino terminus are
nism such as that in F1FoATP synthase. recognized by the signal recognition particle,
which permits transport of the nascent protein
to its new destination in an organelle. Once it
S enters into the new location, this set of amino
acids is removed by proteolysis.
S-adenosylmethionine (SAM) – A condensation prod- single-strand binding protein – Protein binding to
uct of ATP and methionine that functions as a separated DNA strands, such as at the replication
one-carbon donor. fork of DNA, that enables replication (see replica-
saccharide – Sugar (Latin). tion fork).
saturated – Fatty acids containing no double bonds. soft nucleophile – An electron-rich center that is less
saturation – A high and unchanging enzyme velocity localized and more easily polarized.
achieved at high substrate concentration. species hierarchies – The classification of plants and ani-
Schiff base – An adduct formed between an aldehyde and mals into classes, orders, families, etc.
an amine. splice variants – RNA products that differ despite arising
secondary metabolism – Pathways that produce special- from the same transcription event due to differ-
ized metabolic products (secondary metabolites) ences in how the exons are joined together.
that are not essential for the physiologic function spontaneous – A term that has diffuse meanings in ther-
of the organism. modynamics; often a substitute for exergonic,
secondary metabolites – Molecules that are end prod- but at other times used to mean nonenzymatic. It
ucts of pathways specific to one or a few organ- is not a well-defined thermodynamic term.
isms and, thus, are less universal than primary spontaneous generation – The erroneous hypothesis
metabolic routes. Secondary metabolites pro- that living organisms arise from nothingness.
tect the organisms from being consumed by src – Sarcoma virus protein (src is the sarcoma virus gene),
predators. it is the parent of a family of protein kinases that
secondary structure – A regular, repeating structural lead to phosphorylation of tyrosine residues,
element of an amino acid that is formed when called src kinases.
a segment of the molecular chain folds in three src homology 2 – A protein-building domain in the src
dimensions. (sarcoma virus like) family of cytoplasmic tyro-
semialdehyde – A molecule containing a dicarboxylic sine kinases, which can bind phosphotyrosine-
acidand an aldehyde. containing proteins.
semipermeable – A membrane that permits the trans- S6K (S6 Kinase) – A protein target of the small subunit
port of molecules but acts as a barrier to others; of the ribosomal assembly that is a substrate of
partially permeable. mTOR.
Glossary xli
stacking interaction – An attractive force between each ATP by direct chemical means, such as phospho-
hydrogen-bonded base pair (AT and GC, the glycerate kinase or succinyl CoA thiokinase.
“rungs” of the double helix “ladder”) above and sugar acid – A monosaccharide derivative in which the
below them along the long axis of the DNA dou- carbonyl group is oxidized to an acid, such as gly-
ble helix (see double helix). ceric acid.
state – A thermodynamic condition in which certain suicide substrate – An inhibitor that is converted by the
variables, such as temperature, pressure, volume, enzyme into a molecule that either binds tightly
and number of moles, are fixed. or covalently and impedes further enzymatic
state variables – Variables that determine the state of a reaction with true substrates.
system, including temperature (T), pressure (P), supercoils – Circular or closed loops of DNA that occur
volume ( V), and number of moles (n). when DNA is twisted around its own axis, which
statins – Inhibitors of hydroxyl-methyl-CoA reductase, changes the number of turns in the double axis
the rate-limiting step of cholesterol biosynthesis, (see double helix).
these drugs are used to lower blood cholesterol supersecondary structure – In protein molecules, the
concentration particularly in low density lipo- combinations of α-helices and β-sheets con-
proteins (LDLs). nected through loops that form folding patterns
statistical thermodynamics – A derivation of the ther- stabilized through the same types of linkages as
modynamic principles that uses the laws of large in the tertiary level.
numbers, calculating the behavior of atoms and surfactant – An amphiphile acting at an air–water
molecules on the basis of statistical laws. interface.
stator – The stationary part of a motor mechanism (see surroundings – Everything in the universe apart from
rotor). the system under study.
steady-state – A condition in which the intermediates Svedberg – A unit of measure equal to 10 −13 seconds used
of a process, such as a metabolic pathway, are as a measure of size of large macromolecules and
constant with time, while the pathway substrate particles. The term refers to the material’s sedi-
decreases and pathway product increases. mentation during ultracentrifugation.
steady-state assumption – In enzyme kinetics, the symmetrical intermediate – Within some enzyme
assumption that the intermediates of a reac- mechanisms that are rearrangement reactions,
tion mechanism are constant with time (see such as isomerases, addition of protons and/or
steady-state). electrons lead to an intermediate that displays
steady-state constant (Km) – A ratio of rate constants symmetry. The second half of the mechanism
for an enzymatic reaction, the constant is also will then be a mirror image of the first.
the concentration of substrate corresponding to synthase – Enzymes that catalyze joining reactions that
the half-maximal initial velocity for an enzyme. do not involve a high energy intermediate mol-
stereochemistry – The arrangement of atoms of a molecule like ATP. Synonym: lyase (named for the
ecule in space. reverse direction).
stroma – Water space in the chloroplast between the synthetases – See ligases.
membrane of the chloroplast organelle and the system – That portion of the universe under study.
thylakoid membrane.
strong acid – An acid that completely dissociates in
water, forming H+ and an anion. T
strong base – A base that dissociates completely in water,
forming OH- ion (lowering H+ ion concentration) TATA – Located in the promoter region of most eukaryotic
and a cation. genes, a consensus sequence (5'-TATAAAA-3') at
subcutaneous fat – Less saturated lipid-containing adi- −25 nucleotides upstream of the site of the initia-
pocytes found just under the skin. tion of transcription.
substrate – Reactants for enzyme reactions or pathways. telomerase – An enzyme that forms, maintains, and
substrate cycle – Reactions that involve the same path- repairs the ends of chromosomes (see telomeres).
way substrates and products, but in opposing telomeres – The repetitive nucleotide segments located at
directions. Together, the reactions catalyze a net the ending segments of chromosomes; these pro-
hydrolysis of a high energy phosphate intermedi- tect the chromosome from deterioration.
ate such as ATP. These cycles enable more precise temperature – The energy measure of a system that
metabolic control (also termed futile cycle). shows the average kinetic energy of its molecules.
substrate-level phosphorylation – Reaction(s) that form termination – Occurs when the RNA reaches a specific
a high energy phosphate intermediate such as sequence (terminator) and the transcription
xlii Glossary
complex dissociates from the DNA, thereby transhydrogenase – Enzyme that catalyzes movement of
releasing the RNA polymerase to begin a new electrons between NADH and NADPH.
initiation event (see transcription, termination transketolase – Transferase enzyme that uses thiamine
sequence). as a bound cofactor to exchange carbon frag-
termination sequence – In RNA transcription, a poly- ments between substrates, converting one ketone
merase-specific DNA binding protein found close into another.
to the end of a coding sequence; rho-independent transfer RNA – Form of RNA that binds a triplet codon
sequences are inverted and form a hairpin back in mRNA and carries an amino acid to incorpo-
onto the sequence; rho-dependent terminators rate the next unit in a protein chain.
rapidly unwind the DNA–RNA hybrid formed translation – A cellular process in which messenger RNA
during transcription, thereby freeing the newly (mRNA) provides a template for protein synthe-
synthesized RNA (see transcription). sis to form new protein.
tertiary structure – The entire three-dimensional translocase – An enzyme catalyzing membrane trans-
arrangement, or conformation, of a protein chain port, i.e., a transport protein.
in three dimensions. transporter – A protein enabling membrane transport;
tetrahydrobiopterin – Redox cofactor involved in the synonymous with translocase.
hydroxylation of phenylalanine to tyrosine. trehalose – The disaccharide glucose (α1→α1) glucose
tetrahydrofolate – Cofactor involved in one-carbon found in bacteria, fungi, plants, and insects.
metabolism. tricarboxylic acid cycle – see Krebs cycle, citric acid
thermodynamics – A field of study of energy changes cycle.
of processes useful in understanding chemical triose – A three-carbon monosaccharide that is the
reactions. smallest sugar.
thermophiles – Organisms that thrive at very high tem- triosephosphate isomerase (TIM) domain – A domain
peratures. They are part of a larger group of that is donut-shaped, with an inner portion
extremophiles. composed of a β-sheet and an outer portion of
thioredoxin reductase – Enzyme catalyzing the reduc- α-helices (see domain).
tion of thioredoxin, which is involved in forming triosephosphate isomerase – A glycolytic enzyme that
deoxynucleotides. The mechanism of thiore- catalyzes the interconversion of two isomeric
doxin reductase is the same as that of glutathione three-carbon phosphorylated sugars.
reductase. type I diabetes – A disease in which blood glucose
through-space effect – An electrostatic interaction concentrations are above normal due to little
within a molecule that involves interaction or no insulin production by the pancreas (for-
between attached groups, such as the carboxyl merly known as juvenile or insulin-dependent
and amine groups of an amino acid. diabetes).
thylakoid membrane – Most interior membrane of the type II diabetes – A disease in which blood glucose con-
chloroplast; separates the inner (lumen) space centrations are above normal due to tissue insen-
from the stroma. sitivity to insulin (formerly known as adult-onset
titration curve – A plot of pH versus the equivalents of a or noninsulin-dependent diabetes).
strong base (or strong acid) in a solution of a weak
acid (or weak base).
topoisomerases – A class of enzymes that catalyze U
changes in double-stranded DNA by transiently
cutting one or both strands of the helix; in super- ubiquitin – A small protein that can become attached
coiling the twisting results in a shortened mol- to cytosolic proteins and serve as a signal for
ecule (see supercoils, double helix). degradation in the proteasome (see proteasome,
transaldolase – Transferase enzyme that exchanges ubiquitination).
carbon fragments between substrates using the ubiquitination – The process of attachment of ubiquitin
same mechanism as aldolase. to a protein (see ubiquitin).
transaminase – Enzyme catalyzing exchange between umami receptor – Taste receptor responsive to gluta-
an amino acid and a ketoacid. mate, responsible for the savory sensation.
transcription – The synthesis of ribonucleic acid (RNA) uncompetitive inhibition – A reversible inhibitor that
from a deoxyribonucleic acid (DNA) template. binds exclusively to the enzyme-substrate com-
transferases – A category of enzymes that catalyze reac- plex (also termed anticompetitive inhibition).
tions that move a piece of one substrate on to uncoupling – The dissociation of ATP formation (phos-
another. phorylation) from mitochondrial electron
Glossary xliii
transfer. Uncouplers accelerate electron trans- Vmax/Km – The first order rate constant for an enzymatic
port but diminish ATP formation (see coupling). reaction as substrate concentration approaches
universe – All that exists; in thermodynamics, the sum of zero.
the system and surroundings.
unsaturated – Fatty acids containing double bonds.
Examples: arachidonic acid, oleic acid. W
upstream – In transcription, the sequence before the
start site (see downstream). warfarin – Synthetic analog of coumarin, an inhibitor of
ureido – The NH2 CO–NH– group, present in citrulline Vitamin K-dependent carboxylation. The drug
and urea. is used to diminish blood clotting, popularly
uricotelic – Organisms using uric acid as their major known as a “blood thinner”.
nitrogen excretion product. weak acid – An acid that only partially dissociates in
ureotelic – Organisms synthesizing urea as their major water. Examples: acetic acid, oleic acid.
nitrogen excretion product. weak base – A base that only partially dissociates in
water. Examples: ammonia, diethylamine.
wobble hypothesis – A proposal explaining how a spe-
V cific transfer RNA molecule can use different
codons in a messenger RNA template, but carry
vectorial – A process that involves both a quantity and the same amino acid. The third base of the trans-
a direction, such as the movement of protons fer RNA anticodon does not have strict base pair-
across the mitochondrial membrane during oxi- ing rules.
dative phosphorylation. work – An energy of motion (displacement), the product
vibrational – A form of elementary energy arising from of force and distance.
the relative motion of two bonded atoms. To a
first approximation, this energy arises from sim-
ple periodic motion. X
visceral fat – Adipose deposits located in the organs,
especially of the abdomen (also termed organ fat, X-ray crystallography – A method for determining
intra-abdominal fat). the structure of a crystal (including proteins
VDAC – Voltage dependent anion channel. Named for an and nucleic acids) by subjecting them to x-ray
in vitro activity, this is an opening of the outer radiation, measuring the scattering pattern
mitochondrial membrane that allows small mol- that results, and mathematically constructing a
ecules up to 10,000 D to cross this membrane. three-dimensional image from that data.
Only proteins are blocked (see porin). xenobiotic – Any substance (often, a drug) introduced
vitalism – The principle (erroneous) that living systems into an organism that is foreign (xeno) to that
do not obey the same chemical principles as inert organism.
materials.
vitalist – One who believes (erroneously) that living sys-
tems do not obey the same chemical principles as Y
inert materials.
vitamins – Precursors to enzyme cofactors, classified as ylid – A resonance-stabilized carbanion involved in
fat or water soluble molecules needed in small nucleophilic addition leading to subsequent
amounts. decarboxylation, as in pyruvate decarboxylase
vitamin B12 – Vitamin involved in methyl transfer reac- and the pyruvate dehydrogenase complex.
tions, having a central cobalt ion (also known as
cobalamin).
Vitamin K – Vitamin used in enzyme post-translational Z
modification to incorporate carboxyl groups that
serve as extracellular calcium binding sites. zwitterions – Molecule having no overall charge contain-
voltage – See potential difference. The term is also used ing balanced negatively charged and positively
for the units of potential difference. charged parts (German word zwitter, meaning a
Vmax – A kinetic constant that is the rate of an enzymatic hybrid of two forms).
reaction at saturating substrate concentration.
Foundations 1
Biochemistry is the study of the chemical nature of biology. Biochemists use chemical ideas
and tools to elucidate living systems. We begin with a review of some chemical concepts:
reactions, kinetics, equilibrium, steady-state, and energy. Next, we introduce acid–base and
redox reactions. Some foundational ideas of biology complete the chapter: cell theory, evolu-
tion, and species hierarchies.
1.1 ORIGINS OF BIOCHEMISTRY
Compared to its component sciences, biochemistry itself is a young discipline. Hoppe-Seyler

coined the word Biochimie in 1877. He also edited the first biochemistry journal, Biological
Chemistry, still in existence today. In this period – the later part of the 19th century – both
the notions of spontaneous generation and vitalism were dispelled, clearing the way for a
discipline that required new thinking.
According to the hypothesis of spontaneous generation, living systems arose from noth-
ingness, as bacteria seem to do in a nutrient-rich broth. In the 1860s, however, Louis Pasteur
demonstrated that bacteria exist in the air; no bacteria appear in the broth if the container
is isolated from the atmosphere. This discovery led to the cell theory, which holds that cells
are the fundamental unit of living systems and arise from other cells. Despite this advance,
Pasteur himself was a vitalist, believing that living systems do not obey the same chemical
principles as inert objects.
Two challenges to vitalism bracketed the work of Pasteur. In 1828, Wohler discovered
that urea could be synthesized in a laboratory from ammonia cyanate. The strictly in vitro
synthesis of an organic compound did not need the “aid of a kidney” as Wohler put it (see
Box 1.1). In 1897, the German chemists (and brothers) Eduard and Hans Buchner showed
that fermentation occurs in extracts from ruptured cells, thus dispelling the notion that cel-
lular organization is required for processes that occur in living systems.
In the 20th century, biochemistry was dominated first by organic chemistry, as metabolic
pathways were discovered, then by enzymology, then bioenergetics, and later by molecu-
lar biologists as the study of DNA intensified. As biochemistry plays an increasingly large
role in physical and chemical sciences today, these disciplines overlap considerably. What
remains distinctive about biochemistry is the chemical perspective of biological phenomena.
Biochemical principles are presented in the following chapters. In the present one, we con-
sider some fundamental concepts of chemistry and biology.
1.2 SOME CHEMICAL IDEAS
To determine if you might need a review or can instead skip to the next section, take the fol-
lowing self-test:
◾◾ Without specifying a value, what is the meaning of Avogadro’s number?

◾◾ Distinguish between atoms, electrons, molecules, and moles.
◾◾ When is it appropriate to use mole units as opposed to grams?
1
2 1.2 Some Chemical Ideas
Box 1.1 Word Origins: Organic
Among its rich meanings, the word organic also implies a sense of the whole, as in organ-
ism, which also implies animate, living, or vital. The root word stems from the Greek ergon,
meaning work in the sense that an organism is a collection of working entities. Vitalists
identified substances believed to be produced only by a living organism. After vitalism
was discounted, organic was redefined rather than cast aside. Today, organic chemistry is
defined as a branch of chemistry that focuses on compounds of carbon. Strictly speak-
ing, inorganic chemistry refers to molecules containing other elements. In recent times,
another meaning for organic has emerged that is closer to its historical roots. In this con-
text, organic is a label for farming methods that do not use synthesized chemicals (e.g.,
fertilizers and pesticides for plants, antibiotics and hormones for animals). Thus, growing
plants and raising animals in this way is said to produce organic food. Whether this is a
definite health benefit is debatable. For example, the absence of pesticides can lead to
greater bacterial content in food. Moreover, there is no settled agreement on exactly
what organic farming is, so products vary. Despite the various uses of the word, we are
concerned with only the definition of organic chemistry as the chemistry of carbon
compounds.
◾◾ Why is the equilibrium constant for a reaction expressed as the equilibrium concen-
trations of the products multiplied together, divided by the equilibrium concentra-
tions of the substrates multiplied together?
◾◾ How does equilibrium relate to kinetics?
The ideas of the mole, Avogadro’s number, atoms, and molecules are presented in Appendix
A.1. Here, we consider here the notions of kinetics and thermodynamics and other chemical
principles with the assumption that those ideas are well in hand.
1.2.1 REACTIONS AND THEIR KINETIC DESCRIPTION
Suppose substances A and B react to form substances C and D. This is a generic reaction,
which can be written in the form:

(1.1) A + B C + D
A and B are called substrates; C and D are products. Each represents a chemical species and
can also be called a compound or a metabolite. To visualize what is happening, let us relax
our molecular thinking and describe the molecules pictorially as Figure 1.1. In this represen-
tation, the reaction involves removing a piece of molecule A and affixing it to B, thus creating
C and D. This is a mechanistic view. To more fully characterize the reaction, we consider
each direction separately. Fixing a direction allows us to define the rate of a reaction, a char-
acterization known as kinetics. Consider first the forward direction, which proceeds from
left to right. This reaction is written:

(1.2) A + B ® C + D
FIGURE 1.1 A generic reaction.

Chapter 1 – Foundations 3
FIGURE 1.2 Collision theory of reaction rates.
Suppose there are 3 A molecules and 4 B molecules, as shown in Figure 1.2. Each A can com-
bine with any of 4 Bs. This procedure follows Equation (1.2), which excludes the reaction of A
with itself or B with itself. There are 12 possible collisions (3 × 4). Another way of expressing
the situation is that the reaction is proportional to 3 × 4. This analysis is formally known as
collision theory: the rate of a reaction is proportional to the number of collisions. Since the
number of molecules of a substrate is represented by its concentration in solution, the rate of
our reaction is proportional to [A]×[B].
The rate in the forward direction we write as ratef. It can be expressed as an equality rather
than a proportionality by introducing a constant, known as the rate constant (see Box 1.2).
The equation that results is:
(1.3) ratef = k f [A][B]
where k f is the rate constant for the reaction in the forward direction. The overall rate is
measured as a change in concentration per unit time (typically in seconds).
For the reverse reaction, C + D → A + B, following a similar line of reasoning, we arrive at
a similar expression:
(1.4) rater = k r[C][D]
The terms in this expression are rater, representing the rate in the reverse direction of our
original equilibrium of Equation (1.1), and kr, the rate constant in this direction. The expres-
sions for reaction rates shown in Equations (1.3) and (1.4) can be generalized to any reaction.
While independent of changes in concentration, the rate constant can vary with environ-
mental conditions, such as temperature or ionic strength.
A rate, by definition, has only one direction. Yet chemical reactions are commonly writ-
ten with double arrows between substrates and products as in Equation (1.1). This notation
indicates that both forward and reverse reactions can occur and is commonly used when a
reaction has reached a position of equilibrium.
1.2.1.1 EQUILIBRIUM
Perhaps the most fundamental idea in chemistry is that of equilibrium. A state of equilib-
rium exists when the forward and reverse rates of a reaction are equal. Once reactions reach
equilibrium, no further observable change occurs in substrate or product concentrations
4 1.2 Some Chemical Ideas
Box 1.2 Rate Constants
A rate constant is neither a rate nor a concentration. Rather, it is merely a constant of pro-
portionality between the rate of a reaction and the concentrations. The constancy refers
to its independence of concentration. Rate constants are symbolized by a lower case k,
and have units that vary with the reaction itself. Since the rate is in units of concentration
per time, say mol/l/sec, and concentrations are in units of mol/l, then the rate constants
must have units that balance. Thus, in the example used in the text, the rate depends
on two compounds, so that the rate constant is second-order, and has units of M−1sec−1
included in the table below:
Reaction Order Rate Constant Units

Aconst → products 0th or zero-order M sec−1
A→ products 1st sec−1
A + B → products 2nd M−1sec−1
A + B + C → products 3rd M−2sec−1
The first entry in the table represents a situation in which the reaction is entirely inde-
pendent of substrate concentration. First-order reactions are common when the reactant
is in very high concentration and thus does not appreciably change with time over the
reaction course. It is more common in enzymatic reactions, which we consider in a later
chapter.
Notice the pattern of the rate constant units, which shows how they are sequentially
divided by the concentration as the order increases. It is important to recognize this vari-
ation to firmly identify the rate constant term that represents a proportionality that is
independent of concentrations.
unless there is a change in external conditions. Reactions that have not yet achieved equilib-
rium have a driving force toward the equilibrium situation, just as a rolling ball eventually
comes to rest as the forces acting on it balance. To become familiar with the state of equi-
librium, we examine the origins of the equilibrium expression and the equilibrium constant.
As a definition of the equilibrium condition, the rate of the forward reaction equals the
rate of the reverse reaction, that is:

(1.5) ratef = rater
Since the forward and reverse rates were expressed by Equations 1.3 and 1.4, we can make
these substitutions to arrive at:

(1.6) k f [A][B] = k r[C][D]
Sometimes, to emphasize the fact that we are describing an equilibrium situation, subscripts
are added to the concentration terms (e.g., they appear as [A]eq, [B]eq, etc.). We will assume
the concentrations expressed are those at equilibrium under our conditions. Rearranging:

(1.7) k f /k r = [C][D]/[A][B] = K eq
where Keq is the equilibrium constant. This equation defines this constant in terms of equi-
librium concentrations and shows its relationship to the rate constants. It is apparent from
our derivation that the reaction components at equilibrium represent a balanced state of the
collisions in each direction.
Another statement of this type of balance is known as the Le Chatelier principle. For a
reaction at equilibrium, this principle states that concentrations will shift to counter changes
in the concentration of any component. While this principle is simply a restatement of the
relationship between concentrations and the equilibrium constant (Equation 1.7), it has an
intuitive value. Suppose in our reaction that [A] is increased; we might imagine an experi-
menter adding in more A. This increase will push the reaction from left to right until the
original Keq value is obtained. The idea of an increase in the concentration on one side push-
ing the reaction to the other side is apparent from the mathematical requirements for rates
and equilibrium as developed here. However, the Le Chatelier principle can intuitively pre-
dict how the reaction components change to re-establish an equilibrium.
Equilibrium can also exist in the case of multiple connected reactions, such as:

(1.8) A B C D E
Under biological conditions, though, multiply connected reactions require a different model:
the steady-state.
1.2.2 THE STEADY-STATE
One problem with applying the equilibrium model to living cells is that biological systems
never reach equilibrium. Thus, a more elaborate system must be used to model metabolism:
the steady-state. A sequence of connected reactions can be said to achieve a steady-state
when the concentrations of all reaction intermediates are constant. Nonetheless, the first
substrate may be depleted with time, and the final product accumulates. To understand this
concept, we can compare it to an equilibrium, which requires a minimum of two compo-
nents, as in this example:

(1.9) S P
and we can characterize it with the equilibrium constant:

(1.10) K eq = [P]eq /[S]eq
To describe a steady-state, we need a minimum of 3 components, for example, a substrate (S),

an intermediate (I), and a product (P):

(1.11) S ® I ® P
Equation (1.11) represents two reactions:

(1.12) S ® I

(1.13) I ® P
just as an equilibrium has two reactions, a forward and reverse reaction. The similarity also
extends to the rates of the reactions in both models. In our steady-state example, the rates
of these reactions are also equal. The distinction is that the steady-state produces a net flow.
Over time, [S] decreases, [I] is constant, and [P] increases. In biological systems, there are
usually multiple intermediate species between S and P. In Equation (1.14), for example:

(1.14) S ® I ® J ® K ® L ® P
all of the intermediate rates (e.g., S I, I J, etc.) are equal. Not only that, the overall rate, S P is
the same too. The result is that the intermediate concentrations (I, J, K, and L above) do not
6 1.3 Acid–Base Reactions
FIGURE 1.3 The steady-state window. People are moving through a building at a constant
rate. As long as the entry and exit rates are equal, the number of people visible in the large win-
dow will be constant (eight). At any instant in time, the eight people in this intermediate state
will be different.
vary with time: they are the ones forming the steady-state. The intermediates are constant
because each one is formed at the same rate at which it is removed.
The concentrations of S and P need not be constant, however. To achieve a constant rate
of S → I – a necessity for the steady-state – there are two possibilities. First, another reaction
may supply S at the same rate as it is converted to I. Alternatively, [S] may be saturating for
the first reaction, meaning that its concentration is so high that its depletion is negligible. The
product P cannot revert to L, so its accumulation does not affect the rate of L → P.
Suppose the metabolic pathway of Equation (1.14) achieves a steady-state. We can
form an analogy by considering a line of people forced to move single file through a gate.
Figure 1.3 shows people flowing through a building without backup. The number of people
entering represents the [S]; the number of those leaving represents the [P]. At any time we
glance at the window in this building, we always see eight people. Of course, if we look at
another time, the people will be different, but their number is the same. This situation is the
essence of the steady-state.
While the steady-state is distinct from an equilibrium, equating the two is a common sci-
entific error. An equilibrium is often used for situations where there is an element of balance,
not recognizing that this is inappropriate when a series of chemical reactions produce a net
flow. In that situation, only the steady-state model is appropriate.
Finally, it is important to stress that both equilibrium and steady-state are models. Thus,
while they are usually appropriate for the situations we will encounter, they are always
approximations. In some instances, they are wildly inaccurate ones. For example, before an
enzymatic reaction or series of reactions can form constant intermediate concentrations,
neither model applies. This condition is the pre-steady-state, which requires a different
model entirely.
1.3 ACID–BASE REACTIONS
The idea of acid–base chemistry is at once familiar and poorly grasped. As it permeates our
study of biochemistry, it is worth detailed study. We will introduce reactions involving acids
and bases in the next chapter (Water) as we are most interested in their behavior in water,
the cell’s solvent. Here we explore the origins of acids and bases and why a seemingly simple
concept can be elusive.
Acids (meaning sour) and bases (meaning bitter) are each represented among the five taste
receptors on the tongue. Citrus fruits are everyday examples of acids; what is literally called
citric acid is a specific molecule, an intermediate of the Krebs cycle (Chapter 10). A classical
base is formed by mixing ash from burned wood with water (“potash” or potassium hydrox-
ide). The problem of the scientific classification of acids and bases is not a simple one.
In fact, there are three separate views of acid–base chemistry. The first is the Arrhenius
definition, which defines an acid as a substance that increases the proton concentration of
a water solution. A base is a substance that decreases the proton concentration of a water
solution. Apart from the restriction that this requires water as the solvent, it is also not easy
to distinguish features within the molecule that contribute to acid or base properties. Still, it
allows us to classify molecules with no protons, such as carbon dioxide, as an acid.
The second view is the most prominent in this book: the Bronsted definition. A Bronsted
acid is a proton donor, and a Bronsted base a proton acceptor. Often, this definition is stated
without the Bronsted adjective. A proton must be a part of the acid, in this view, with the
ability to release the proton into solution.
A third acid–base description is the Lewis definition. This view is the most general but
more subtle. Here, the focus is on electron pairs rather than protons. An acid is defined as
an electron pair acceptor. A base is defined as an electron pair donor, arising from lone pair
(that is, nonbonding) electrons. This is the broadest definition; for example, we can consider
metal-ligand chelates as acid–base pairs.
While this brief introduction to acids and bases is long on definitions and short on details
and examples, we will expand this notion throughout our study of biochemistry. We will
encounter numerous acid–base reactions. While most of our focus will be on the Bronsted
definition, the expansion to the others will pay dividends in our ability to understand all
types of acid–base reactions.
1.4 REDOX
Apart from acid–base chemistry, the other pillar of biochemistry involves redox reactions.
Redox is a contraction of reduction-oxidation. There are many parallels between acid–base
and redox reactions. There are multiple views of redox, and once again, we need to consider
different viewpoints to become familiar with the notion.
The first definition of redox is an oxidation model that considers that a molecule becomes
more oxidized as it literally incorporates oxygen atoms. The other partner of the reaction
then becomes more reduced. This model is somewhat limiting, as it requires that oxygen be
a part of the molecule. Still, it remains widely used in the field of pyrotechnics (fireworks).
More practically, for our purposes, it allows us to easily rank oxygen-containing organic
molecules as increasingly oxidized as they contain more bonds to oxygen, as in the series
illustrated in Table 1.1.
However, it provides no insight into the process and ignores the fact that redox reactions –
like acid–base reactions – must occur in pairs. One partner is oxidized, the other is reduced.
The second definition of redox is useful when ions can exchange electrons with other
ions. This is the basis of redox chemistry first discovered by Alessandro Volta in 1800, who
TABLE 1.1 Increasing Incorporation of Oxygen

Progressively Increases Oxidation State of Molecules
Molecule Structure Oxidation state of C
Alkane −3
R CH3
Alcohol −1
R CH2OH
Aldehyde O +1
R C
H
Acid +3
R COOH
CO2 O C O +4
8 1.5 Energy
discovered the battery (and is honored in the unit volts). The Volta battery is a redox reaction
that exchanges electrons in an overall reaction that is represented by the oxidation half:

(1.15) Zn ® Zn 2 + + 2e-
and the reduction half:

(1.16) Cu 2 + + 2e- ® Cu
By definition, oxidation is a loss of electrons, and a reduction is a gain of electrons. The two
are coupled; combining both halves produces the overall reaction:

(1.17) Zn + Cu 2 + ® Zn 2 + + Cu
A third definition is necessary because the organic molecules of biochemistry are not gen-
erally redox-active ions and are not always readily discerned by their oxygen atom content.
Thus, a more general redox definition is needed: an artificial assignment of an oxidation
number to each atom in a molecule. We will consider this model in more detail in later
chapters, but the essence of the assignment is to consider which atom across a bond attracts
electrons more strongly (i.e., is more electronegative) and assign a number as if that atom
was a pure ion. These numbers are the same in actual ions: in Equation 1.15, for example,
the Zn species has an oxidation number of zero, and the Zn2+ has an oxidation number of 2.
The assigned oxidation numbers of the carbon atom explicitly shown in the middle column
are given in the last column of Table 1.1. The method for calculation will be presented in
Chapter 8.
1.5 ENERGY
Energy is a notion that is at once simple and complex. It is simple because it is part of every-
day vocabulary: a child knows about “having a lot of energy,” the relative difficulty of walk-
ing uphill, and the existence of friction. It is also complicated. Energy exists in multiple
forms: electrical, gravitational, pressure-volume work, and heat flow, each having specific
conditions and equations. An important observation from the early 19th century was that all
forms of energy are interconvertible. Further advances in that century led to a formal science
of energy changes known as thermodynamics, which can elucidate the energy of a waterfall,
a galloping horse, a chemical reaction, or that contained within a single molecule.
There are two faces of thermodynamics: the classical and the statistical. The classical
approach uses a few postulates and definitions but makes no assumptions about the exact
nature of the systems under investigation – even the existence of molecules is unnecessary.
The statistical approach considers the collective behavior of large numbers of molecules.
Both approaches lead to accurate descriptions of energy changes. To begin our study of ther-
modynamics, we need to consider three of its fundamental ideas:
internal energy, enthalpy, and entropy.
Internal energy is the energy of a system, the sum of the work and heat transferred to that
system. Work is an energy of motion, such as the product of force and distance. Heat is
an energy transfer due to a difference in temperature between a system and its surround-
ings. Enthalpy (derived from the Greek enthalpos, meaning “putting heat in”) is the heat
absorbed or released by a system at constant pressure. As this situation is common in labora-
tory experiments – and in living cells – tables of enthalpy changes for reactions are widely
used in chemistry and biochemistry.
Entropy is best understood using the statistical approach to thermodynamics. It is related
to the number of ways that energy can be distributed as a result of a process (usually a reac-
tion). For example, if we suddenly apply brakes in a speeding car, the energy of motion is
FIGURE 1.4 The global CO2 cycle. Radiation from the Sun is used to energize electrons in
the chloroplasts of plants, forming the high-energy molecule NADPH. These electrons drive
the synthesis of sugar from atmospheric CO2 in plants; this overall process is photosynthesis.
Subsequently, the plants are eaten by animals, and the sugars (and other molecules) break down
to release CO2 in to the atmosphere, completing the cycle. In the breakdown process, energy is
trapped as electrons in NADH and used to produce ATP for cell processes.
redistributed into particles of tire rubber on the street, as well as frictional heat. Entropy
increases with an increase in energy dispersion.
When we discuss energy in a biochemical context, we are invariably referring to a com-
bination of enthalpy and entropy changes, called free energy. The free energy change for
a reaction determines whether it can proceed in the direction written. A more expansive
treatment of thermodynamics, free energy, and the relationship between free energy and
equilibrium are discussed elsewhere in this book. Here, we will just assume that energy is
equivalent to free energy.
We commonly speak of both molecules and portions of molecules as having high energy.
Two high energy molecules of particular importance in biochemistry are nicotinamide
adenine dinucleotide (NADH) and adenosine triphosphate (ATP). Both of these molecules
are mobile cofactors that allow communication between hundreds of different reactions
within the cell. NADH transfers electrons; we consider it a donor of high energy electrons.
ATP transfers a terminal phosphoryl group; we consider this molecule to be a high energy
phosphate compound.
The connection between electron flow and ATP formation is a profound one in biochem-
istry. In the global energy cycle (Figure 1.4), radiation from the sun induces the formation of
high energy electrons by driving the formation of nicotinamide adenine dinucleotide phos-
phate (NADPH), a close analog of NADH. NADPH then donates electrons to convert atmo-
spheric CO2 to sugars and other molecules. Subsequently, animals consume those sugars,
moving their electrons to NADH, and using this to form ATP. In the process, sugars and
other molecules form CO2, which is once again utilized by plants.
1.6 CELL THEORY
The cell – the smallest unit of life – is a key organizing principle in biology. Single-cell organ-
isms, such as bacteria, yeasts, and protozoans (e.g., the paramecium), comprise the largest
number of species. Our primary focus, however, is on multicellular organisms, primarily
humans.
10 1.6 Cell Theory
The features of a representative mammalian cell are illustrated in Figure 1.5, which shows
internal organelles and their arrangement within a cell. There are many variations on this
theme, which we can illustrate by a few examples. At one extreme, the mammalian red blood
cell or erythrocyte in humans contains no intracellular inclusions (organelles), retaining
only the plasma membrane and cytosol. Another extreme is the fat cell or adipocyte, which
has many typical organelles, but also an exaggerated lipid droplet that comprises virtually
the entire cell volume, appearing as a featureless blob with a thin film of cytosol between
the fat droplet and the plasma membrane. While not shown in the representative cell of
Figure 1.5, lipid droplets are found in many cells as multiple small spherical organelles rather
than the single large one of the adipocyte. As another variation, melanosomes are modified
lysosomes that contain the dark brown colored compound melanin, present in the epider-
mal cells of the skin’s outer layer. Melanin absorbs ultraviolet light, protecting the underly-
ing tissue from radiation damage. Finally, Figure 1.6 shows a section of a skeletal muscle
cell. The regular appearance of stripes (striations) represent protein components responsible
FIGURE 1.5 The mammalian cell. A representative drawing of the cell shows the compart-
ments that are created by membranes. The cell itself is separated from its exterior by a plasma
membrane; several interior membranes, such as the mitochondria, the lysosome, and the endo-
plasmic reticulum, define separate reaction spaces within the cell.
FIGURE 1.6 A skeletal muscle cell. A transverse section of a stained skeletal muscle cell is illus-
trated, demonstrating the large number of protein fibers filling the cytosol. The various organ-
elles fit between groups of the protein fibers. Source: https://commons.wikimedia.org/w/index
.php?curid=29092851
FIGURE 1.7 The cell as a concept. The cell is represented as separate chemical reaction spaces,
separated by semipermeable membranes. Specific exchanges across the membranes are indi-
cated by the arrows drawn across the membranes in both directions.
for contraction (actin and myosin fibers). Some organelles are visible such as mitochondria,
stained as dark purple spheres between fiber groups.
Despite wide variation in the arrangements of cells, there is a functional unity to their
behavior in terms of biochemistry. As represented in Figure 1.7, we can conceptually draw
a few key compartments that are responsible for most metabolic activities in cells. An outer
plasma membrane encloses the cell, serving as its boundary with the outside. This mem-
brane, like all cell membranes, is semi-permeable. Some molecules readily cross the mem-
brane, such as O2, H2O, and CO2. Others can traverse the membrane only if there is a specific
carrier protein they can pass through. From a chemical viewpoint, the membranes create
separate water spaces to isolate chemical reactions. Only those molecules that can com-
municate across these spaces – those that can diffuse or those with a specific transporter –
can participate in the reactions. The major water space within the cell is called the cytosol.
Other membrane-delimited organelles that define separate water spaces for reactions are the
endoplasmic reticulum, the mitochondria, and the nucleus. In addition to defining reac-
tive spaces, membranes are sites of lipid biosynthesis.
1.7 SPECIES HIERARCHY AND EVOLUTION
Evolution is a fundamental principle of biology. It is also commonly misunderstood, perhaps

due to the popularization of the phrase “survival of the fittest”. It is ironic that this phrase
has survived in the popular lexicon but did not find itself in the landmark work on evolution,
Charles Darwin’s On the Origin of the Species.
Darwin formulated the theory of evolution based on the extremes of plant and animal
species found in the isolated Galapagos Islands, 1000 km off the coast of Ecuador. The idea
was that species arose from other species, and those that could adapt to their environment
survived and reproduced. Eventually, adaptive characteristics emerged. It is now common-
place to apply evolutionary principles to biochemistry, mapping the formation of numerous
enzymes and DNA sequences from early origins to divergent present forms.
To catalog the complexity of the immense number of known species, currently believed to
number in the tens of millions, scientists have long turned to another important biological
organizing principle: the classification hierarchy. At the top of the currently accepted hierar-
chy are three domains: Archaea, Bacteria, and Eukarya (see Box 1.3: Archaea). There are
several lower levels of organization: Kingdom, Phylum, Class, Order, Family, Genus, Species.
Our focus is on mammalian, mostly human, biochemistry; although plant, bacterial, and,
occasionally, yeast examples will be considered. Most discoveries in biochemistry are based
on only a very small sampling of the biological universe, largely due to practical consider-
ations. Still, there are many indications of similarity of pathways and mechanisms between
12 1.8 Biological Systems
Box 1.3 Archaea
It was the archaea that forced a revision of the previous standard taxonomy of organisms.
Archaea – like bacteria – are single-celled and have no nucleus but constitute a separate
domain from bacteria (now also called true bacteria or eubacteria). They do have other
organelles, like mitochondria. The categories of Archaea, Bacteria, and Eukaryota are
domains, classifications above the kingdom.
The more intriguing aspect of the archaea is that it includes the extremophiles, organ-
isms that can survive what we would consider harsh conditions. For example, extreme
temperatures, such as hot springs, harbor archaea species as do ocean depths (extreme
pressure) and areas of extremes of acid or base. There are also the radiophiles, organ-
isms that survive intense radiation (both ultraviolet and ionizing radiation). Adaptations
include modifications to lipids for the membranes of the organisms, unusual repair
enzymes for their DNA, and even specialized “antifreeze proteins” to survive extreme
cold. As a practical and perhaps most famous example, one enzyme isolated from a spe-
cies of the hot springs Archaea, a DNA polymerase, is the basis of method of amplification
of DNA, which uses cycles of heating and cooling to greatly amplify the amount of DNA
present and has moved beyond the research laboratory to become a standard forensic
tool to detect extremely low DNA concentrations.
different organisms. Some differences between organisms will be considered to get a sense of
how biological variation is expressed.
1.8 BIOLOGICAL SYSTEMS
There is another hierarchy that identifies the viewpoint of the investigator of biological sys-
tems. Figure 1.8 shows a ranking with organism at the top level and subatomic particles at
the bottom. There are even higher levels than the organism, such as a population, which have
medical and scientific importance (as in the transmission of infection between organisms).
However, levels beyond the organism are usually of interest to other fields, such as the social
sciences.
The rankings in Figure 1.8 can also be used to exemplify two extremes in the analysis of
biochemistry: a view close to the top, taking in as wide a swath as possible, is called holistic.
Those who favor the holistic view argue that it leads to physiologically relevant observa-
tions. And a view close to the bottom, examining specific interactions at a molecular or
submolecular level, is called reductionist. Those who favor the reductionist view argue that
it alone allows firm conclusions to be drawn because the number of variables studied is much
smaller and better defined than in the holistic approach. Both are essential, and we will move
between them in the text. We begin with a reductionist approach, examining the chemical
properties of water, the molecule most essential for life.
FIGURE 1.8 Hierarchies of biological study. Different levels of investigation are possible in the
experimental study of biology. Physiologists usually study higher levels, such as organisms, organ
systems, and organs. Physicists typically study atoms and subatomic particles. Biochemists usu-
ally examine the intermediate levels. If the study is closer to the top of the hierarchy, it is consid-
ered physiologically relevant, and the approach is holistic. However, there is greater uncertainty
about the data. If the study is closer to the bottom, it is considered more exact, and the approach
is reductionist. However, the relevance to biology is less certain. Both approaches, while in con-
flict, are essential for a full understanding of biology.
KEY TERMS
adipocyte extremophiles
Arrhenius definition free energy
Bronsted definition heat
cell theory high energy electrons
collision theory high energy molecules
cytosol high energy phosphate
electronegative holistic
endoplasmic reticulum internal energy
energy kinetics
enthalpy Le Chatelier principle
entropy Lewis definition
epidermal melanosomes
equilibrium mitochondria
14 Bibliography
nucleus spontaneous generation

oxidation number steady-state
rate constant thermodynamics
reductionist vitalism
saturation vitalist
semipermeable work
BIBLIOGRAPHY
M.G. Ord, L.A. Stocken, Eds. Foundations of Modern Biochemistry, Vol. 1, Early Adventures In
Biochemistry. JAI Press, Greenwich, CT, 1995.
P.S. Cohen, S.M. Cohen. Part of a multi-volume history of biochemistry; the information in the pres-
ent chapter is found mostly in the first two chapters. Wohler's Synthesis of Urea: How do the
Textbooks Report It?, J. Chem. Educ., 73 (1996) 883–886.
H.J. Morowitz. Discussion of the details of the Wohler synthesis and the slow rejection of vitalism
despite the finding. Entropy for Biologists. An Introduction to Thermodynamics. Academic
Press, New York, 1970.
Kotz, J.C., P.M. Treichel, J.R. Townsend, and D.A. Treichel. A gentle introduction to thermodynam-
ics with biological examples. Acids and Bases: The Arrhenius Definition. In Chemistry and
Chemical Reactivity, Instructor's Edition, Vol. 116. 9th ed. Cengage Learning, Stanford, CT.
2015.
I. Yajima, L. Larue, The Location of Heart Melanocytes Is Specified and the Level of Pigmentation in
the Heart May Correlate with Coat Color, Pigment Cell Melanoma Res. 21 (2008) 471–476.
G. Raposo, M.S. Marks, Melanosomes: Dark Organelles Enlighten Endosomal Membrane Transport,
Nat. Rev. Mol. Cell Biol., 8 (2007) 786.
B. Lewin, L. Cassimeris, V.R. Lingappa, G. Plopper, eds. Melanosomes are considered a lysosome
related particle. Cell. Jones and Bartlett Publishers, Boston, 2007.
L. Marulis, K.V. Schwartz, A cell biology textbook providing broad introductions to life at the cellular
level. Five Kingdoms. An Illustrated Guide to the Phyla of Life on Earth. 2nd ed. W.H. Freeman
and Co., New York, 1988.
J.A. Coker, In addition to a catalog displaying drawings and photographs of examples of the major
life forms, the work contains an introduction to the science of taxonomy, the hierarchies of life.
Recent Advances in Understanding Extremophiles, F1000Res, 8 (2019).
F.C. Fang, A. Casadevall, Reductionistic and Holistic Science, Infect. Immun., 79 (2011) 1401–1404.
Smith, M.B. Organic Chemistry: An Acid-Base Approach. CRC Press, Boca Raton. 2011.
Water 2
We are mostly water. Our daily weight fluctuations are mainly due to the gain or loss of water
from our bodies. At a philosophical level, most feel an almost mystical connection to water.
Thus, Ishmael’s claim in Moby Dick that we all yearn for the sea stirs a sentiment in readers
well over 150 years after it was written.
In most cells, the relative number of water molecules is even more impressive than its
preponderance by weight. The number-dominance of water molecules is a consequence of its
low molecular mass (18 daltons). In fact, for every single protein molecule, there are 75 lipid
molecules, 100 Na+ ions, and 20,000 water molecules.
Although we generally refer to the molecule itself as water, each physical phase has its
own name: water for the liquid, steam for the gas, and ice for the solid. While abundant,
water is curiously distinct from other liquids. One unique property of water is its ability to
dissolve most other substances. While it falls short of being a “universal solvent”, the num-
ber and type of substances that dissolve in water are important biological considerations.
Water also has unusually strong heat retention compared to other liquids. Finally, water has
unusual phase transitions, such as a very high boiling point, and unusual behavior at low
temperatures. For example, the solid form of water is less dense than the liquid form. All of
these properties can be traced to the molecular structure of the water molecule.
2.1 STRUCTURE OF WATER
To begin our examination of water, we consider the isolated molecule as it exists in water
vapor. The phases of water are illustrated in Figure 2.1, which also indicates two separate
groupings. The gas and liquid are called fluid phases. Molecules in these phases are mobile
and distribute evenly throughout space. Liquid and solid phases are called condensed
phases. Molecules in these phases are considered incompressible: molecules cannot be
brought appreciably closer together by increasing pressure, for example. The overlapping of
characteristics demonstrates that phase properties depend upon context. A study of each
phase, and the transitions between them, provides insight into both the behavior of water
and its interaction with other molecules.
2.1.1 GAS PHASE WATER
The essential feature of the gas phase is that there is virtually no interaction with other
molecules. Physiologically, gas phase water is important for both animals and plants. For
example, the transition of surface water to water vapor is a cooling mechanism called evapo-
rative heat loss.
The properties of biochemical consequence arise from the arrangement of electrons and
nuclei within this molecule. It is evident from the formula H2O that two atoms of hydrogen
are attached to a single atom of oxygen, and that the molecule is electrically neutral. When
the atoms are drawn on paper as they are known to be arranged geometrically into the two-
dimensional structure of Figure 2.2. The H–O–H bond angle is 104.5°. The underlying reason
for this structure becomes apparent when we consider the three-dimensional representation
of Figure 2.3. The oxygen atom contains eight electrons: two inner electrons (which are not
15
16 2.1 Structure of Water
Phases
Gas
Fluid
Liquid
Condensed
Solid
FIGURE 2.1 Liquid water is a condensed fluid. The three phases of matter can be character-
ized as fluid or condensed (incompressible). The liquid state of water (as with other substances)
shares the properties of being both fluid and condensed.
H 104.5° H
FIGURE 2.2 Two-dimensional structure of water. The three atoms of water can be drawn in
a plane; the two bonded hydrogens make an obtuse angle, the value of which can only be
explained by a consideration of its three-dimensional structure.
FIGURE 2.3 Three-dimensional structure of gas phase water. Four orbitals of the water mol-
ecule are shown; water has a tetrahedral structure. (a) Space-filling diagram shows bonded H
orbitals coming out of the page, and nonbonded electron pairs behind the plane of the page. (b)
Tetrahedral orientation depicts the oxygen in the center of the tetrahedron and the nonbonded
orbitals and bonded hydrogen atoms at the vertices.
involved in bonding) and six valence electrons. The number of valence electrons corresponds
to the Period number or column heading in the Periodic Table (Figure 2.4).
In the water molecule, 6 valence electrons of oxygen mix with two from the H atoms,
recombining into four molecular orbitals, arranged as a tetrahedron, as in Figure 2.3. The
tetrahedral structure is the result of the mutual repulsion of the electrons of these orbitals.
Because they are all attached to a central oxygen, and thus in forced proximity, the orbitals
assume a geometrical arrangement where they can be as far apart from one another as pos-
sible. The two lone-pair (unbounded) orbitals have a somewhat greater negative charge and
repel each other more than the orbitals with bonded electrons. As a result, the H–O–H angle
is somewhat less than the 109.5º of a perfect tetrahedron, as in methane (CH4).
2.1.2 PARTIAL CHARGES AND ELECTRONEGATIVITY
A covalent bond is, by definition, a sharing of electrons between the nuclei of two atoms,
but the sharing is usually unequal. In the case of the single bond between O and H in water,
Chapter 2 – Water 17
FIGURE 2.4 An abbreviated periodic table. The periods I through VIII are shown without the
traditional intervening transition elements. Those elements towards the right and the top have
the greatest electronegativity. Only the elements N, O, and F (shaded) participate in hydrogen
bonding; in organic molecules, only N and O form significant hydrogen bonds. Elements of bio-
logical importance are highlighted in blue.
the electrons are located closer to the oxygen atom. This uneven sharing results in partial
charges that can be assigned to each atom involved in the bond. The O atom is partially nega-
tive and the H atom is partially positive.
In general, atoms differ in their inherent ability to attract the electrons that are involved
in bonding. The ability of an atomic nucleus to attract electrons in the bond to itself is called
electronegativity. Values of electronegativity can be directly assigned to each atom, based
upon measurements of bond energies involving different pairs of atoms. A normalized scale
runs from zero (no electronegativity) to four (greatest electronegativity). A clear trend in
electronegativity values is present in the arrangement of the elements in the Periodic Table.
Those top right elements are the most electronegative; those in the bottom left are the least
electronegative. These periodic trends indicate the different powers atoms have in attracting
the bonding electrons. Elements on the right side are more stable when they attract electrons,
completing their outer shell. Elements towards the top of the table have fewer electrons to
shield the valence electrons from the charge of the nucleus. The electronegativity values of
several elements are listed in Table 2.1.
We are now in a position to examine the electronegativity values for the O–H bond in
water. Since hydrogen has a value of 2.1 and oxygen a value of 3.5, the electronegativity dif-
ference is 1.4. It has been estimated that this makes the bond about 39% ionic. This merely
adds quantification to the previous point that the electrons in this bond are more strongly
attracted to the O than to the H. Thus, while the bond is in one view covalent, it is in fact a
polar bond, or a partially ionic bond.
TABLE 2.1 Electronegativity
The polar bond is represented by a vector. This vector is shown as a directed line segment
drawn with the origin at the H and the arrowhead at the O. The vector may be further deco- Values of Selected Elements
rated with a little plus sign at the origin to denote the relatively positive (electron deficient) Element Electronegativity
part of the distribution. F 4
After assigning vectors for both of the bonds in water in three dimensions, it is possible O 3.5
to take a vector sum (Figure 2.5). Because the molecule has a nonzero net sum, it has a net N 3
polarity. The two orbitals of oxygen that are not bonded (containing electron pairs only) do C 2.5
not contribute to the overall polarity of the molecule. Not all molecules with polar bonds S 2.5
have a net polarity; for example, CO2 has polar bonds, but the vectors directly oppose each H 2.1
other, so the molecular polarity is zero (Figure 2.6). Cl 3
Because water has a net polarity, it is a polar molecule. We can assign partial charges Na 0.9
to the atoms within the molecule. By convention, polarity is indicated by the Greek letter
O
+ +
H H
Net vector sum
FIGURE 2.5 Net polarity of water. The vectors resulting from electronegativity differences are
indicated with plus annotations at their positive ends. The sum of these vectors is the overall
polarity of the molecule.
+ +
O C O
Net sum = 0
FIGURE 2.6 Lack of polarity in carbon dioxide. Despite having electronegativity differences
within the molecule, the sum of the vectors in carbon monoxide is zero, so that it is a nonpolar
molecule.
d–
O
d+ d+
H H
FIGURE 2.7 Partial charges in gas phase water. The differences in electronegativity are indi-
cated by partial positive and negative charges within the water. Thus, the bonding is only partly
covalent.
d– d+
d–
d+
d+
d+
FIGURE 2.8 The hydrogen bond. As a result of the partial charge interactions, two water mol-
ecules are shown interacting through a hydrogen bond. As indicated by the dotted circle, the
bond requires one hydrogen atom attracted to two oxygen nuclei at once. The dotted portion
of the bond is identified as the hydrogen bond.
“delta”, superscripted with a negative or positive sign to symbolize the partial negative or
positive charges as in Figure 2.7. These features of the water molecule in the gas phase give
rise to the unusual interactions between molecules that occur in the condensed phases and
provide an explanation for the distinct properties of water.
2.1.3 CONDENSED PHASE WATER: HYDROGEN BONDING
Just as people change their behaviors in social settings, molecules in the condensed
phases – liquid and solid – display new properties. Molecules in such close proximity are
incompressible , which means that external pressure cannot cause them to move perceptively
closer together. Most of our interest will be in the liquid state. Still, some consideration of
the solid phase water, as well as the transitions between states (phase changes), will enhance
our understanding of water.
How neighboring water molecules are held together is illustrated for two water mol-
ecules in Figure 2.8. The hydrogen atom between the two oxygen nuclei in the figure has
Box 2.1 Covalent and Ionic: Discreet or Continuous Distinction?
In your first encounters with chemistry, a bond is defined as being either ionic or covalent.
The division is important in distinguishing chemical behavior. For example, an ionic solid
has a much higher melting point than a solid of a covalently bound compound, because
of the strong adhesion of a large network of opposite charges. Yet ionic compounds dis-
solve readily in water and separate completely, losing the ordered structure they had as
a crystal and becoming isolated ions surrounded by water. When organic compounds
melt, only intermolecular bonds are disrupted, so they have far lower melting points and
retain their identity in solution. It would seem that covalent and ionic bonds are therefore
entirely distinct. Yet, we have noted that the electrons in a covalent bond involving dif-
ferent atoms are not equally shared and give rise to partial charges. An ionic bond is one
extreme where the electrons are so skewed that one atom is essentially fully negative
and the other fully positive; a covalent bond involving the same atom on each side of the
bond is the other extreme. Between these extremes is a continuum from ionic to degrees
of ionic bonding to covalent. Still, we must be cautious in thinking that all chemical situ-
ations can be considered as a continuum: the fundamental changes of quantum physics
occur in discrete steps. However, bonding characteristics are a macroscopic quality that
appears as a continuous process involving partial ionic bonds. We could consider water,
for example, as a compound of O2− associated with two H+ ions such that the molecule
has a preponderance of negative charge on one side and a preponderance of positive
charge on the other. Because of the incomplete dissociation of water in its liquid state,
we would conclude that water is not actually an ionic compound but rather a partially
covalent one.
one bond indicated as a solid line and the other as a dotted line. The solid line represents
a covalent bond, whereas the dotted line represents a hydrogen bond, one of the most
important interactions in biochemistry. The bridged hydrogen atom has its only electron
shared in the covalent linkage. Therefore, the hydrogen bond is essentially an electrostatic
attraction of the hydrogen nucleus to one of the lone electron pairs of the oxygen atom
of the other water molecule. Box 2.1 offers a further discussion of covalent and ionic
bonding.
Covalent bonds are much stronger than hydrogen bonds. The average bond energy for
a covalent O–H bond is 467 kJ/mol, whereas a hydrogen bond is in the order of 10 kJ/mol.
Despite the relative weakness of individual hydrogen bonds, they become a dominant force
that explains the peculiar properties of water in the aggregate. The three-dimensional view
of Figure 2.9 applies to both water and ice. The latter is the regular crystal from which
the figure is taken. The liquid state is sometimes considered to be a “flickering crystal”;
the distinguishing fluid property, adjusting to the shape of any vessel, is conferred by the
greater motion of the liquid water molecules and their ability to exchange positions in
the liquid crystal-like structure. Another way to view the liquid state is that the structure
resembles the crystal but has an occasional defect that allows water molecules to rearrange
themselves.
Molecules in the solid state usually assume a denser structure than they do in the
liquid state. For example, the density of liquid ethanol is 790 kg/m 3, while the solid form
density is 950 kg/m 3. This difference is typical: molecules are usually closest together in
a crystalline solid, and an increase in temperature (needed to convert a solid to a liquid)
tends to cause the molecules to move further apart, decreasing the density. Liquid water,
however, is already highly ordered, and the small amount of motion at temperatures
close to the freezing point causes water molecules to move closer to one another on the
average than in ice. As ice melts to water, its density increases, reaching a maximum
at 4°C. At higher temperatures, the increased motion does reduce the density of liquid
water (Figure 2.10). Thus, water has a maximum density at 4°C, which explains why ice
floats on water.
FIGURE 2.9 Condensed phase water. Two views of condensed phase water are shown. (a) The
oxygen atoms are shown to bond to four positions throughout the structure. (b) A “side view” of
condensed phase water showing a hexagonal structure that results from a plane of water mol-
ecules in regular array. In ice, these structures are representative. Liquid water approximates this
structure but continuously breaks and re-forms bonds so that the structure overall is in constant
change, even though most of it appears as an ordered structure.
FIGURE 2.10 How the density of water varies with temperature. The density of water has a
maximum at 4oC. The variation of density with temperature is shown over a range of (a) zero to
30o C; (b) zero to 10oC. The change is modest but significant. There are two opposing effects as
energy (temperature) is increased: a closer approach of more active molecules and increased
separation as the temperature is increased further.
2.1.4 HYDROGEN BONDING IN CONDENSED PHASE WATER

TABLE 2.2 Boiling Points
of Common Liquids We have just seen that the unusual solid to liquid transition of water is a consequence of its
Fluid BP °C hydrogen-bonded structure. The unusually high boiling point of water is also a consequence
Water 100 of hydrogen bonding (Table 2.2).
Benzene 80 The extensive hydrogen bonding in water produces cohesiveness that can resist the escape
Carbon Tetrachloride 77 tendency of molecules into the gas as the temperature is increased. Thus, a higher tempera-
Chloroform 61 ture (and correspondingly greater energy) is needed to boil water.
Ethanol 79 Another way of thinking about water is to view it as a hydride of oxygen. Other hydrides of
closely related atoms are HF (hydrogen fluoride), H2S (hydrogen sulfide), and NH3 (ammonia).
TABLE 2.3 Boiling

Points of Hydrides
Hydride BP °C
H2O 100
HF 20
NH3 −33
H2S −61
FIGURE 2.11 Comparing water to similar structures. Three-dimensional structures of HF, H2O,
H2S, and NH3 indicates that NH3 and H2S are very similar to water, yet do not exhibit water’s exten-
sive hydrogen bonding due to geometry differences (NH3) and lack of sufficient electronegativ-
ity differences (H2S).
HF does not occur biologically; H2S occurs as a product of bacterial respiration. Ammonia
is an intermediate in mammalian metabolism and is excreted as a final waste product of
nitrogen metabolism in fish. The boiling points of these substances are listed in Table 2.3.
Two factors – electronegativity and molecular geometry – explain the striking differences
in boiling points between water and the other polar hydrides. The electronegativity of F is
greater than the electronegativity of O (see Table 2.1). HF has a considerably lower boiling
point than water due to a difference in spatial arrangement. Three-dimensional views of
these molecules are shown in Figure 2.11.
We have already seen how water interacts with other water molecules, creating an exten-
sive network. It is not possible for either HF or NH3 to form extended hydrogen-bonding
networks; they can only form limited intermolecular hydrogen bonds. Oxygen and sulfur
are in the same column of the Periodic Table, so one might expect hydrogen sulfide to have
properties very similar to those of water. However, the S–H bond is insufficiently polar; S
and H have similar electronegativities. As a result, hydrogen sulfide (or any –SH-containing
molecules) does not form hydrogen bonds. Accordingly, H2S has an extraordinarily low boil-
ing point compared to H2O. Indeed, in our everyday experience, both H2S and NH3 occur
only as gases.
Thus, water’s uniqueness stems from its combination of electronegativity differences and
the geometry of its hydrogen-bonded interactions. These features lead to a high degree of
ordering and extensive lattice formation that is not shared by other very similar molecules.
This combination of properties is the key to understanding how water interacts with other
substances. At one extreme are molecules that strongly interact with water and tend to dis-
solve in water. At the other extreme are molecules with virtually no interactions with water.
The hydrophobic effect explains this, a concept discussed in the next section.
2.2 THE HYDROPHOBIC EFFECT
The aphorism “oil and water don’t mix” can be readily understood by considering the proper-
ties of the condensed state of water. Consider the molecule decane:
CH 3 - CH 2 - CH 2 - CH 2 - CH 2 - CH 2 - CH 2 - CH 2 - CH 2 - CH 3
It is composed entirely of C—C and C—H bonds. The electronegativity of the C–C bond
is zero (2.5–2.5), which is always the case when the same element occurs across a bond.
The electronegativity of the C–H bond is 0.4 (2.5–2.1). Even this small net polarity is
offset by the geometry of the structure, as the polarity vectors (pointing to the carbon
chain) largely cancel each other (see Figure 2.12). Hence decane and compounds with
similar chemical composition are nonpolar and do not form hydrogen bonds with water.
Another nonpolar substance is the oil in oil-and-vinegar salad dressing. It is a common
experience that the dressing settles into separate layers even after they have been vigor-
ously mixed.
22 2.2 The Hydrophobic Effect
FIGURE 2.12 Polarity of methylene residues. Using the vector sum method, the polarity of
the C–H bonds is low, as the small C–H electronegativity difference is further diminished as the
vectors nearly cancel each other out spatially.
The finding that nonpolar molecules separate from water is called the hydrophobic effect
(Box 2.2). This term should be taken literally: an effect rather than an accurate description of
the underlying chemistry. Although hydrophobic means “water-hating”, alkanes, such as dec-
ane, do not hate water at all. In fact, decane has a stronger affinity for water molecules than
it has for other decane molecules! The water–alkane attraction results from water inducing
a dipole in the electrons of the alkane. The induced dipole attraction, however, is a minor
factor. Instead, our focus should be on water itself: water prefers to interact with other water
molecules rather than with lipids. Hydrophobicity is sometimes described as water squeez-
ing out nonpolar molecules so that the water can form its own phase. For a more thorough
consideration of the forces underlying the hydrophobic effect, see Box 2.3.
Box 2.2 Word Origins: Hydrophobic
The word hydrophobic was used as a medical term for hundreds of years as a synonym for
the disease rabies. A consequence of late-stage rabies infection is an inability to swallow,
so those afflicted were said to have a “fear of water”, or hydrophobia. The use of “hydro-
phobic” for the exclusion of water from nonpolar substances is more recent, stemming
from the early part of the 20th century. Even though the scientific usage is well-established
and there is no debate about what it means, its strict translation implies that chemically
water and lipid molecules display repulsive forces, which is not the case. The term only
describes how water and nonpolar substances form separate phases as if they had some
antipathy for each other. Ironically, as an archaic medical term for rabies, it is far removed
from the underlying pathology. As an embedded term for water–nonpolar interactions, it
is similarly a description of the phenomenon, far removed from the underlying chemistry.
Box 2.3 A Thermodynamic Look at the Hydrophobic Effect
In our discussion of the hydrophobic effect, we considered the formation of two phases,
colloquially an oil and a water phase, and suggested that the principal forces were water
interactions with itself rather than with the oil. While this is true, the underlying reason
for it is somewhat subtle and requires that we apply the ideas of thermodynamics.
In particular, the free energy of a process is determined by two factors: the enthalpy
and the entropy. To apply these notions, consider the process of moving from one
state to the next. Suppose our process moves individual lipid molecules (substrate) into
coalesced lipid molecules (product). Studies of nonpolar molecules in water have shown
that hydrogen bonding of water molecules still occurs when lipid molecules are forced
into a water solution; a representative drawing of two such molecules in a water solu-
tion is shown in Figure B2.3a. The binding energy (enthalpy) of water arranged around
lipid molecules is similar to the binding energy of water molecules in a solution of pure
water. Thus, enthalpy is not the driving force for the hydrophobic effect. The “product”
represents the change modeled in Figure B2.3b, where the two lipid molecules have
coalesced, and a cage of hydrogen-bonded waters surrounds both of them.
The placement of water molecules around the lipids is believed to be far more ordered
than in pure water; alternatively, we can say that water in this cage has less freedom of
FIGURE B2.3 Hydrophobic Effect: Box Figure. (a) Two nonpolar molecules, introduced into
water, become individually surrounded by hydrogen-bonded water molecules. The enthalpy
of these water molecules is similar to that of the bulk water (not shown, but assumed as back-
ground in the figure). However, the entropy is less; these waters are more ordered than bulk
water. (b) The same molecules are now shown, but now the lipid molecules are associated
with each other. The number of surrounding water molecules is much less than that in (a),
which means the unfavorable ordering of water molecules has been reduced. Compared to
the situation in (a), the entropy is greater, and the situation in (b) is more favorable from a free
energy standpoint.
movement. This increase in order observed in Figure B2.3a translates into a decrease in
the system entropy, which is an unfavorable free energy change. When the lipid mole-
cules coalesce in Figure B2.3b, there is considerably less ordering of the water molecules.
This result is evident in the figure, as fewer water molecules are required to corral the
more extensive lipid combination shown in Figure B2.3b than the individual ones shown
in Figure B2.3a. Thus, the relatively greater entropy of water drives the association of lipid
molecules together.
2.3 MOLECULES SOLUBLE IN WATER

The bonding between water molecules in pure solutions of water is extensive. The ability of
another molecule to dissolve in water requires displacement of water–water indent hydrogen
bonds by a stronger interaction.
Imagine you are trying to dissolve table salt (NaCl) in water. In the solid form, NaCl
is a crystal, a regular array of interspersed Na+ and Cl−. There is a considerable difference
between Na (0.9) and Cl (3) electronegativities, so electrons are not shared equally between
these ions. In the crystal, Na+ bears a full charge of +1, Cl− bears a full charge of −1, and the
attraction is purely electrostatic. This attraction is ionic bonding.
When solid NaCl is added to water, its identity disappears just as white table salt disap-
pears as we stir it into water. Solubilization occurs as the Na+ becomes surrounded by the
partially negative portion of water molecules and the Cl− becomes surrounded by the par-
tially positive portion of water molecules, as illustrated in Figure 2.13.
The dissociation can be represented as a reaction, in which solid NaCl is converted to
isolated Na+ and Cl− ions that are surrounded by water (hydrated). It is customary to write
the hydration as:
(2.1) NaCl ® Na + + Cl -
24 2.4 High Heat Retention: The Unusual Specific Heat of Liquid Water
FIGURE 2.13 Solubilizing NaCl. Entry into solution involves water molecules giving up their
own hydrogen-bonded interactions in favor of an ionic interaction between the partial charge
of the oxygen atom of water to the sodium ion and the partial charge of the hydrogen atom of
water to the chloride ion. Thus, solubilized NaCl has a different and separate identity from the
solid.
Water is omitted from this chemical equation because it is not strictly a reactant or product
in the formal sense. Still, the reaction is impossible without it. What is happening is that the
energy of hydration – the strength of interaction of the ions for water – overcomes both the
strength of interaction of water for other water molecules (hydrogen bonding), as well as the
NaCl lattice interactions (ionic bonding), and the ions are brought into solution.
We can assess the tendency for a solvent to bring crystals like NaCl into solution by
exploring the dielectric constant. This constant is a measure of the ability of a solvent
to separate charged species (detailed in the addendum to this chapter). There is a correla-
tion between a high dielectric constant and the ability to dissolve ions; typical values are
illustrated in Table 2.4. Common solvents are listed in order of their boiling points. The
table also lists the net dipole (measured as a dipole moment, essentially the net vector of
molecular polarity as illustrated in Figure 2.5), the value of the dielectric constant, and the
solubility of NaCl in that solvent. It is apparent that water has one of the highest dielectric
constants and is best at solubilizing NaCl. Most organic solvents have a dielectric con-
stant an order of magnitude lower; hexane is typical. There is virtually no solubilization of
NaCl in organic solvents. However, Table 2.4 also shows that other factors are responsible
for solubilization. For example, formic acid and dimethyl formamide (DMF) have similar
dielectric constants but differ markedly in their ability to dissolve polar molecules. It is
also clear that while we have seen that water has a permanent dipole, reflected in a signifi-
cant dipole moment, other molecules with higher dipole moments have poor solubilities
for NaCl.
It is the unique geometry of the water molecule that enables it to form strong polar bonds
to salts and allows it to dissolve NaCl readily. This extends to polar molecules that do not
form ions, such as alcohols and certain amines. For example, water forms solutions with
ethanol in all proportions, which is also true of formamide. In all cases of dissolution, the
dissolving molecules displace the hydrogen-bond network of water with stronger bonding
interactions with the solute.
2.4 HIGH HEAT RETENTION: THE UNUSUAL

SPECIFIC HEAT OF LIQUID WATER
Another feature that results from hydrogen bonding is an exceptionally high specific heat for
liquid water. The specific heat is the amount of energy needed to increase the temperature of
1 g of a substance by 1°C. Effectively, the high specific heat of water means that water acts as
TABLE 2.4 Solvent Properties

BP Dipole Moment Dielectric Constant NaCl Solubility
Name Structure (°C) (debyes) (dimensionless ratio) (g/100 ml)
Formamide O 210 3.7 109 9.4
HC
NH2
DMF O 153 3.82 47.2 0.04
H 3C CH
N
CH3
Water O H 100 1.85 80 36
Formic Acid H 100 1.41 58 5.2

C
HO O
Acetonitrile N 81 3.92 36.6 0.003

C
H 3C
Hexane 69 0 2 <0.001
Methanol CH3OH 68 1.7 33 1.4
Acetone O 56 2.88 20.7 <0.001
H 3C CH3
a “temperature buffer”; that is, heat loss or gain is particularly slow compared with other sub-
stances. This stems from the strength of the hydrogen bonds. As temperature is increased,
the bonds bend but do not break (i.e., water molecules adhere to each other very tightly). Heat
goes into the vibrational energy of the bonds, but the lattice structure resists translational
changes (i.e., water molecules breaking free of the H-bonded lattice). Hence, the temperature
rise is minimal in water. In the solid form (ice), the bending modes are not available so that
this substance does not share the high specific heat of water; ice is more similar to the solid
form of alcohol in specific heat.
When solutes are dissolved in water, as discussed in the prior section, the specific heat of
the solution is less than that of pure water. The higher specific heat of pure water is due to
the regular three-dimensional arrangement of its molecules. In the salt solution, hydrogen-
bonding interactions are disrupted by water interaction with ions, decreasing the tempera-
ture-buffering power of water–water hydrogen bonds.
2.5 IONIZATION OF WATER
Pure water dissociates to a very small extent into ions. The reaction is illustrated in Figure 2.14.
The products appear to arise from the migration of a proton from one water molecule to a
second, creating two ions. The mechanism is diagrammed using a curved arrow, which indi-
cates the movement of an electron pair. The products are the hydroxide ion and a protonated
water molecule. While the protonated water molecule (H3O+) predominates in solution, with
very little of the proton itself (H+), it is customary to represent protonated water as just H+.
Thus, the dissociation equation is written as:
(2.2) H 2O H + + OH -
26 2.6 Some Definitions for the Study of Acids and Bases
FIGURE 2.14 Water equilibrium. Two water molecules are illustrated in the process of ion for-
mation. The electron pair movement shown essentially moves the bond from one O–H group
to its neighbor in a hydrogen-bonded structure. The two ions formed are the hydroxide ion,
OH−, and the hydronium ion, H3O+. The latter is abbreviated in common use, substituting the
simpler H+.
Although the ionization of water takes place to a very slight extent, it is nonetheless an
extremely important topic of biochemistry. With pure water, the dissociation produces an
equal amount of H+ and OH−. However, many substances dissolve in water and alter this bal-
ance, which leads us to the subject of acids, bases, and pH.
2.6 SOME DEFINITIONS FOR THE STUDY OF ACIDS AND BASES
Of the definitions for acids and bases introduced in the first chapter, the simplest is the
Arrhenius definitions that apply in water solutions. These hold that:
◾◾ Acids are species that increase the hydrogen ion concentration when added to water.
◾◾ Bases are species that decrease the hydrogen ion concentration when added to water.
Based on these definitions, we can define neutral, acidic, and basic solutions as follows:
◾◾ In a neutral solution, [H+] = [OH−]

◾◾ In an acidic solution, [H+] > [OH−]
◾◾ In a basic solution, [H+] < [OH−]
Species such as NaCl dissociate completely in water but do not affect the concentration of H+
or OH−. Hence, NaCl dissolves in water to form a neutral solution.
Hydrochloric acid (HCl, the acid in your stomach that helps digest food) and acetic acid
(CH3COOH, the principal component of vinegar) dissolve in water according to the follow-
ing equations:

(2.3) HCl ® H + + Cl -

(2.4) CH 3COOH CH 3COO- + H +
Equation (2.3) for HCl is similar to the dissociation of NaCl in that both species completely
dissolve in water, indicated by the presence of a single forward arrow. Unlike the dissociation
of NaCl, the dissociation of HCl leads to an increase in H+, thus making HCl an acid. The fact
that the reaction is irreversible is expressed by labeling HCl a strong acid.
The dissociation of acetic acid in Equation (2.4) also increases the hydrogen ion concentra-
tion in water and is therefore an acid. Because CH3COOH is only partially dissociated, it is a
weak acid. Like all weak acids, we must express the equation as an equilibrium.
Sodium hydroxide (NaOH) and ammonia (NH3) are bases. These species in water undergo
the following reactions:
(2.5) NaOH ® Na + + OH -
(2.6) NH 3 + H 2O NH 4 + + OH -
Each of these compounds produces OH−. Increasing the [OH−] lowers the [H+] in water since
the equilibrium in Equation (2.2) will be shifted to the left. NaOH dissolves completely in
water, making it a strong base. NH3 only partially dissociates, as evident from the equilib-
rium expression; it is a weak base.
Most of our interest in the field of acid–base chemistry is in weak acids and weak bases. A
final definition involving these allows us to simplify our analysis. Let us reverse the direction
of Equation (2.4) and write:

(2.7) CH 3COO- + H + CH 3COOH
This equation has an equilibrium constant that is just the reciprocal of that for Equation
(2.4). What has changed is that the ionized species CH3COO− is a base. By our definition, it
must lower the [H+] as it proceeds from left to right. To experimentally achieve this result,
we would have to add a salt of CH3COO− such as sodium acetate to water. We would then
observe a decrease in the hydrogen ion concentration of water. However, this base is in a
charged form, making it a conjugate base.
2.7 THE PH SCALE
The pH scale is used to represent the very small numbers that occur for the values of the [H+].
By definition:
(2.8) pH = -log [ H + ]
The term on the right-hand side of Equation (2.8) is the common logarithm (base 10). Some
typical values and the corresponding hydrogen ion concentrations, [H+], are shown in
Table 2.5; a fuller discussion of the mathematics of logarithms is presented in Appendix A1.
The benefit of using pH is its compact representation of the hydrogen ion concentration.
However, there are some clear pitfalls. The pH is a double transformation of the hydrogen ion
concentration: first, it is a negative value, so that increasing pH means fewer hydrogen ions.
Second, it is a logarithm, so that a change of 1 unit of pH means a ten-fold change in [H+]. It
is also important to note that pH, being a logarithm, has no units, even though [H+] has units
of concentration.
To become more familiar with pH, consider the range of the pH function as shown in
Figure 2.15. Scale A shows the range of pH values typically encountered in human body flu- TABLE 2.5 pH and
ids, from a pH of about 2 to about 8. Scale B is the readout of a typical pH meter. This is the [H+] Values
most familiar representation of the pH concept and how we measure it experimentally. In Scientific
fact, Scale B is somewhat generous, as the electrode of this instrument is poorly selective for pH [H+] (M) Notation
protons above a pH of about 12. Scale C is the actual range of the pH function: from negative 5 0.00001 10−5
infinity to positive infinity. It is important to recognize the artificiality of Scale B: these are 7 0.0000001 10−7
merely the markings on the pH meter. For example, the pH of a 2 M solution of HCl has a 9 0.000000001 10−9
negative value (−log 2).
28 2.8 The Henderson–Hasselbalch Equation
FIGURE 2.15 Three pH scales. The range of the pH function depends upon your view-
point. a) In human physiology, the range is about 2 to 8, with some of the key values indicated.
b) Measurements of pH using the pH electrode and meter are typically marked from zero to 14,
giving the popular conception that this is the definition of the range of pH. c) The true range of
the pH function is from negative infinity to plus infinity. The value of 7 appears to be in the center
only because pure water at room temperature and pressure has this value; otherwise, even that
entry is arbitrary.
2.8 THE HENDERSON–HASSELBALCH EQUATION
Most biological acids and bases are weak rather than strong. Consider the dissociation of
acetic acid:

(2.9) CH 3COOH CH 3COO- + H +
The equilibrium expression for this equation is:
(2.10) K a = éëCH 3COO- ùû éëH + ùû / [CH 3COOH ]
in a similar way to the development of equilibrium constant expressions in Chapter 1. Taking

the log of both sides of Equation (2.10):

(2.11) (
log K a = log éëCH 3COO- ùû éëH + ùû / [CH 3COOH ] )
Using the properties of logarithms, Equation (2.11) can be written as:

(2.12) (
log K a = log éëH + ùû + log éëCH 3COO- ùû / [CH 3COOH ] )
In the previous section, we presented the definition of pH:

(2.8) pH = - log éëH + ùû
Analogously, we can perform the same transformation of the equilibrium constant Ka and
introduce the term pKa:

(2.13) pK a = -log K a
which has advantages for representing the equilibrium constant of a weak acid in a similar
way to the use of pH in place of [H+]. For example, consider the two representations of the Ka
for the dissociation of acetic acid:

(2.14) K a = 1.8 * 10 -5

(2.15) pK a = 4.7
Using the definitions for pH (Equation 2.8) and pKa (Equation 2.13), we can make substitu-
tions into Equation (2.12) and rearrange it to arrive at:
(2.16) pH = pK a + log éëCH 3COO- ùû / [CH 3COOH ]
Equation (2.16) is the Henderson–Hasselbalch equation, which is a log transformation

of the equilibrium expression for a weak acid. To demonstrate its utility, consider a sample
problem. Suppose we had a solution of acetic acid with a pH of 7.4 (the value for normal
blood). We can readily determine that most of the acetic acid would be in the salt form, that
is, CH3COO−. Inserting values for the pH and pKa:
(2.17) 7.4 = 4.7 + log éëCH 3COO- ùû / [CH 3COOH ]
so that:

(2.18) log éëCH 3COO- ùû / [CH 3COOH ] = 2.7
Raising each side of Equation (2.18) to the power of 10 (taking anti-logarithms), we get:

(2.19) éëCH 3COO- ùû / [CH 3COOH ] = 102.7 = 500
Equation (2.19) tells us that the concentration of CH3COO− is 500-fold greater than the con-
centration of CH3COOH. It is evident that despite the less than intuitive nature of a negative
logarithm transformation, as discussed in the prior section, using this equation simplifies
calculations. In fact, if we look back at Equation (2.19) and ask the question of which spe-
cies – the salt or the acid – predominates under the conditions stated, it is clear that it is the
salt form. Without knowing the exact number represented by 102.7 we know it is more than
100 (102) and somewhat less than 1000 (103). Thus, answering the qualitative question can be
performed at a glance, using arithmetic only to substrate pKa from pH.
We should appreciate that the answer is an approximation, even with the 500-fold excess.
We conclude that, to a very close approximation, we can assign a charge to the carboxyl
group of −1.
We can use the Henderson–Hasselbalch equation for weak bases as well. As an example,
ammonia can be written in the form of a weak acid dissociation if we start with the conjugate
acid form (as defined in the prior section) and write:
(2.20) NH 4 + NH 3 + H +
In Equation (2.20), the protonated form – NH4+ – is an acid. It is a weak acid, as it is only
partially dissociated, and can be characterized by an equilibrium expression:
(2.21) K a = [ NH 3 ] éëH + ùû / éëNH 4 + ùû
The corresponding form of the Henderson–Hasselbalch equation is:
(2.22) pH = pK a + log [ NH 3 ] / éëNH 4 + ùû
A more general form of the Henderson–Hasselbalch equation that can be used for both acids
and bases is:

(2.23) pH = pK a + log [ X ] / [ XH ]
where X is the base (that is, the less protonated form), and XH is the acid (that is, the more
protonated form). Notice that the use of Equation (2.23) requires that we consider acid–base
equilibria from the standpoint of the Bronsted definition: that is, an acid is a proton donor.
30 2.9 Titration and Buffering
It happens that the vast majority of such reactions in biochemistry adhere to this definition.
It should be noted that, in general, an acid can have two charged forms: zero and negative. A
base can have two charged forms: zero and positive. These are suppressed in Equation (2.23),
but is an important check on our analysis of acid–base behavior and the charge of molecules.
The interaction of pH and molecular charge is a prevalent issue in biochemistry.
2.9 TITRATION AND BUFFERING
The Henderson–Hasselbalch equation provides specific pH values, given the ratio of the
basic and acidic forms of a weak acid and the constant pKa. A titration curve provides a dif-
ferent view of pH as a function of the same ratio. Figure 2.16 shows a titration of acetic acid,
starting with the fully protonated form (CH3COOH) and ending with the fully anionic form
(CH3COO−). In this titration, the strong base NaOH is added in small increments, and the
pH is recorded continuously. Note the relatively flat buffering region centered in the mid-
point of the titration, where pH changes very little as NaOH is added. The center of the buffer
region – where pH changes the least – corresponds to the pKa value. All weak acids and weak
bases are buffers, effectively blunting the response to any added acid or base. The reason for
the behavior can be explained mathematically as follows: when pH = pKa, the Henderson–
Hasselbalch equation reduces to:
(2.24) 0 = log éëCH 3COO- ùû / [CH 3COOH ]
FIGURE 2.16 Acetic acid titration curve. Acetic acid is placed in solution, and NaOH added
until its amount in solution reaches the same number of equivalents, which is moles per charge.
As both species have a unit charge, equivalents and moles are the same in this case. The middle,
relatively flat region shows buffering, that is, a minimal response of pH to an added acid or base.
The center of the buffering region, at 0.5 equivalents, corresponds to the pKa on the pH axis.
Taking the antilog of both sides yields:

(2.25) éëCH 3COO- ùû / [CH 3COOH ] = 1
A small amount of added NaOH will only slightly raise this ratio because substantial and
nearly equal amounts of CH3COO− and CH3COOH are present. Once the concentration of
the two species becomes dissimilar, then the same addition of NaOH will more profoundly
change the ratio. It is common practice to set a limit of a ten-fold excess of one or the other
form and consider this span the buffering range. Perhaps the most significant physiological
buffer is the blood bicarbonate buffer that maintains the extracellular pH in mammals. This
system has two connected equilibria:
(2.26) CO2 + H 2O H 2CO3 H + + HCO3 -
and is maintained by having a relatively high concentration of CO2. Changes in respiration

can adjust the actual concentration of CO2. The concentration of the bicarbonate ion, HCO3−,
is adjusted by the relative retention of the kidney. Note that our definition of acids as species
that increase the hydrogen ion concentration in solution also applies to CO2, which itself has
no protons because it causes an increase in [H+] in solution when it dissolves in water, making
it an Arrhenius acid.
SUMMARY
Water is the most abundant molecule in biological systems and a ubiquitous part of everyday
life. However, compared to other liquids, water has unusual properties. For example, the
exceptionally high boiling point of water reflects intermolecular cohesion as well as its ability
to dissolve a wide variety of polar molecules.
The hydrogen bonding in water contributes to its unusual properties. Hydrogen bonding
is a consequence of the electronegativity difference between H and O, and the geometry of
the water molecule. An extensive hydrogen-bonded network provides the binding strength
that explains the high boiling point. Due to the fixing of molecules in a three-dimensional
lattice, heat energy is stored in the bond vibrations of water rather than in translational
energy, accounting for its high specific heat.
The ready dissociation of water into protons and hydroxide ions, and the ability to
solvate ions, accounts for the ability of water to support the dissociation of acids and
bases. Weak acids, which are partially dissociated, are particularly important biologically.
These can be profitably analyzed using the Henderson–Hasselbalch equation, a logarith-
mic transform of the equilibrium expression for acid dissociation. This equation allows
quick calculations of pH or the ratio of dissociated to undissociated forms of weak acids,
given the pKa. Finally, a full titration of weak acids reveals a buffering region, centered by
the pKa; near this pH value, adding exogenous acids or bases has only modest effects on
solution pH.
REVIEW QUESTIONS
1. Gas phase water has properties similar to most other gases, yet condensed water is
unusual.
a. Why are the properties the same in the gas phase?
b. What are the unique phase transitions of water?
c. What accounts for the unique water properties in general?
2. In a pure water solution, it is possible to calculate the concentration of water itself. What
is its value?
32 Chapter 2 Addendum: The Dielectric
3. Electronegativity values are assigned to individual elements, and yet the concept of elec-
tronegativity applies only to bonds. Explain this disparity.
4. Oxygen and sulfur are both nucleophiles, have the same valence, and form dihydrides.
Yet, H2S does not form hydrogen bonds to other molecules of H2S; indeed, sulfur does
not participate in hydrogen bonding at all. Why is that?
5. Oxygen and nitrogen appear to be very different: oxygen appears in acidic compounds
and tends to assume a negative charge; nitrogen appears in basic compounds and tends to
assume a positive charge. Yet both participate in hydrogen bonding. Explain these facts.
6. Why is it that a molecule like CO2 that contains polar bonds cannot attract polar
molecules?
7. Ammonia is a polar molecule by virtue of its asymmetrical lone pair. However, when
protonated to ammonium, it is now symmetrical. The net polarity of ammonium is zero,
as the polarity vectors sum to zero. Yet, the ammonium ion readily dissolves in water.
Why is this?
8. The hydrophobic effect is due to attractive forces only and not repulsive ones. Rationalize
this with the term “hydrophobic”.
9. A rule of thumb in chemistry is “like dissolves like”. Explain how this applies differently
to polar and nonpolar substances.
10. When enough NaCl is added to water, there is no further dissolution and solid falls out
of solution; this is called saturation. Explain this phenomenon.
11. Water is a “temperature buffer” in that it resists changes in temperature more than other
liquids. Explain this based on water structure.
12. Water ionizes to a very small extent, although this can be increased with increasing
temperature. Provide a reasonable explanation.
13. Protons produced by self-ionization of water have unusually rapid mobility in solution;
they move faster than can be accounted for by diffusion. Provide a possible mechanism
to account for this “proton jumping”.
14. According to the definition of an acid as an entity that increases the proton concentra-
tion in water (known as the Arrhenius definition), we can call CO2 an acid. How could
we handle this situation if, instead, we used the Bronsted (proton donor) definition?
15. While useful, the pH scale can also be confusing because it is a double transform: first,
a log of a very small number, and secondly, multiplied by −1. How does this affect your
intuition about a change in proton concentration?
16. According to Le Chatelier’s principle, a system at equilibrium responds to a change in
one side by shifting the balance towards the opposite side. Thus in A + B ⇌ C + D, an
increase in A will cause the equation to shift to the right side. Explain the basis of this
principle based on the derivation of the equilibrium constant.
17. A salt water solution has a dielectric constant of about half of that of water. What is a
possible explanation?
18. If we add solid bicarbonate of soda (NaHCO3) to water, would this form an acidic or
basic solution? Explain.
19. Describe the dissociation of water in terms of (a) the Arrhenius definition, (b) the
Bronsted definition, and (c) the Lewis definition of acid–base.
20. NaCl is commonly used as a preservative. What would explain this property?
21. We have seen that a conjugate base is the dissociated form of an acid after it has been
introduced into solution. Write an equation for the introduction of a conjugate base in
solution.
22. How can you identify a conjugate acid or a conjugate base in an equation?
23. Assume the pKa of ammonia is 10. At a pH much lower than this, what is the predomi-
nant charge of an ammonia solution? What would the answer be at a pH much higher
than 10?
CHAPTER 2 ADDENDUM: THE DIELECTRIC
While it is true that water has a high dielectric constant and that high values of the dielectric
constant are often correlated to the ability of a solvent to dissolve polar molecules, the dielec-
tric itself is a separate concept from solubility. In order to come to terms with the idea, we
need to start with perhaps the most fundamental equation of electricity, Coulomb’s law. This
law states that the force between two charges is proportional to their product and inversely
proportional to the distance between them:
k * ( q1 * q2 )
F=
r2
where k is a constant, q1 and q2 are the charges, and r is the separation between the charges.
The law was modeled on the inverse square of distance measured for gravity; as a geometric
matter, the area of force lines at a given distance from a point will be proportional to the
square of that distance, as surface area involves a square of the radial distance. The same geo-
metrical concerns also lead to the definition of the constant in Coulomb’s law. As originally
defined in a medium of air, which is similar to the force in a vacuum, it can also be written
for any other medium as:
k ¢ * ( q1 * q2 )
F=
er2
where the constants are redefined, with ε as the dielectric constant appearing in the denomi-
nator. When any medium other than a vacuum separates the charges, the force between
them is diminished. However, this equation is not the whole story because the actual mea-
surement of ε requires a separate concept: capacitance.
A capacitor is a device that historically precedes the storage battery, as it can be used as
an electric storage device. In fact, it was this very device that Benjamin Franklin used (a foil-
lined glass bottle) to connect with a string to lighting to prove that this weather phenomenon
indeed involves electricity. The capacitor has more elaborate uses in electronics; however,
our interest here is the more elementary idea. In Figure 2A.1, the capacitor shown is a pair
of metal plates that are close to each other but not touching. The plates have the same mag-
nitude of charge on them, but the opposite sign; these can represent q1 and q2. To charge a
capacitor, a battery had to be connected to each plate and then removed (the same concept
but more practical than lightning and a wet kite string). In the separation between the plates,
shown in grey, is a material known as a dielectric. Relative to a vacuum, any material will
cause the voltage across the plates to be lower. As voltage is an easily measured property for
this system, it is a simple matter to measure the dielectric constant of the material between
the plates. Water has a fairly high dielectric constant (about 78); that of most organic solvents
are low (about 2). As a practical matter, most dielectrics used in electronics are solids, and
themselves have a fairly low dielectric constant. However, the reason for their use is to enable
more charge on the plates for the same voltage.
When dielectric materials are polar substances, they align with the electric field between
the plates and decrease the voltage across the plates. Thus, the dielectric constant is a rough
measure of polarity, and polar solvents generally dissolve polar solutes. However, this is not
the entire story of solubility. The water molecule geometry and its small size also contribute
to its ability to act as a solvent. To show how the dielectric constant can be divorced from the
ability to dissolve polar solutes, consider the series of solvent molecules Table 2A.1:
The progression of Table 2A.1 shows that solvents’ ability to dissolve polar molecules,
such as the ionic compound NaCl, can be entirely distinct from the dielectric constant. All
three formamide derivatives have higher dielectric constants than water – reflecting their
behavior in an electric field but not how they interact with molecules to dissolve them. The
increased methylation of groups going across the table interferes with hydrogen bonding
and decreases NaCl solubility. (Source: Hernandez et al, Ind. Eng. Chem. Res. 2016, 55, 3,
812–819).
FIGURE 2A.1 A Charged Capacitor.

34 Key Terms
TABLE 2A.1 Formamide Analogs Versus Water: Dielectric and Solubility

Compound Water Formamide N-Methylformamide N-Methylacetamide
Structure O H O O O
CH3 CH3
H H NH2 H NH H 3C NH
Dielectric constant 78 109 182 191

Solubility of NaCl (mol/kg) 6.14 1.55 0.55 .59
KEY TERMS
buffering region
condensed phases
conjugate base
dielectric constant
dipole moment
electronegativity
enthalpy
entropy
fluid phases
free energy
hydrated
hydrogen bond
hydrophobic effect
ionization of water
partially ionic bond
polar bond
polar molecule
strong acid
strong base
translational
vibrational
weak acid
weak base
BIBLIOGRAPHY
J. Burgess, Metal Ions in Solution, Halsted Press, New York, 1978.
J.L.H. Johnson, S.H. Yalkowsky, E. Vitz, A Three-Dimensional Model for Water, J. Chem. Educ., 79
(2002) 1088–1091.
L. Pauling, The Nature of the Chemical Bond and the Structure of Molecules and Crystals: An
Introduction to Modern Structural Chemistry, Cornell University Press, Ithaca, 1960, pp.
393–448.
C. Tanford, The Hydrophobic Effect, Wiley, New York, 1980.
Lipids 3
A defining characteristic of lipids is their relative insolubility in water. Chemically, lipids
are relatively simple – consisting mostly of unreactive hydrocarbons – making this topic
an appropriate next step in our study. Lipids play a role in virtually every aspect of cellular
biology and disease states of the body. The myriad of forms that serve this purpose arises
from relatively modest variations in structure. Here, we consider the major types of lipid
molecules and their principal roles.
3.1 SIGNIFICANCE
The primary functions of lipids are energy storage and membrane formation. Beyond this,
lipids can form micelles in the digestive process, monolayers in the lung lining and lipid
droplets, and serve as signal molecules.
Distinct structures correspond to each function. For the storage role, the predominant
chemical species is the triacylglycerol, also known as triglycerides. These molecules are vir-
tually insoluble in water and are classified as nonpolar lipids. For the membrane function,
the most prominent molecules are the phospholipids, which are partially water soluble.
Membrane lipids are classified as polar lipids. We begin our structural examination by con-
sidering a building block of both triacylglycerols and phospholipids, the fatty acids.
3.2 FATTY ACIDS
Fatty acids are molecules with two parts: a long hydrocarbon segment, called a “tail”, and a
smaller region that typically consists of a carboxyl group, called the “head” (Figure 3.1).
The hydrocarbon tail consists mostly of chemically unreactive methylene groups and a
variable number of double bonds. Table 3.1 presents the structures and nomenclature of
some naturally occurring fatty acids. Most naturally occurring fatty acids have an even num-
ber of carbons, reflecting their biosynthetic origins: they are formed in two-carbon blocks
(Chapter 14).
Fatty acids with no double bonds are saturated, as the carbon atoms have the maximum
number of hydrogen atoms attached to them. The saturated fatty acids listed in Table 3.1
show a regular increase in melting points with increasing carbon chain numbers. The short-
hand notation in the first column of Table 3.1 indicates the number of carbons and double
bonds. For example, butyric acid is a four-carbon fatty acid with no double bonds. Those
fatty acids having eight or fewer carbons melt below room temperature (25°C) and are liq-
uids under normal conditions. Saturated fatty acids longer than this have steadily increasing
melting points and are solids at room temperature. Thus, the fluidity of a fatty acid directly
correlates to its chain length. This is also true of the water solubility of fatty acids; up to about
10 carbons (capric acid), fatty acids are readily soluble in water; beyond this, they do not form
simple water solutions.
Fatty acids containing double bonds are unsaturated, referring to the fact that carbons
of double bonds have fewer than the maximum number of hydrogens attached to them. The
numbers following the colon in Table 3.1 for the unsaturated fatty acids indicates the number
35
36 3.2 Fatty Acids
FIGURE 3.1 Parts of a fatty acid. The hydrophobic portion is the tail; the hydrophilic is the
head. Usually, numbering systems consider the carboxyl group as carbon 1.
TABLE 3.1 Properties of Fatty Acids

Identity Common Name Systemic Name Structure MP °C Source
Saturated
4:0 Butyric Butanoic CH3(CH2)2COOH −5.5 Butter
6:0 Caproic Hexanoic CH3(CH2)4COOH −4 Butter
8:0 Caprylic Octanoic CH3(CH2)6COOH 16 Coconut oil
10:0 Capric Decanoic CH3(CH2)8COOH 31 Coconut oil
12:0 Lauric Dodecanoic CH3(CH2)10COOH 44 Palm, coconut oil
14:0 Myristic Tetradecanoic CH3(CH2)12COOH 52 Milk fat
16:0 Palmitic Hexadecanoic CH3(CH2)14COOH 63 Palm oil
18:0 Stearic Octadecanoic CH3(CH2)16COOH 70 Visceral Fat
Unsaturated
16:1∆9 Palmitoleic Hexadecenoic CH3(CH2)5CH=CH(CH2)7 COOH 0 Macadamia nuts
18:1∆9 Oleic Octadecanoic CH3(CH2)7 CH=CH(CH2)7 COOH 16 Olive oil
18:2∆9,12 Linoleic Octadecadienoic CH3(CH2)4(CH=CHCH2)2(CH2)6COOH −5 Corn oil
18:3∆9,12,15 Linolenic Octadecatrienoic CH3CH2(CH=CHCH2)3(CH2)6COOH −11 Flaxseed
20:4∆5,8,11,14 Arachidonic eicosatetraenoic CH3(CH2)4(CH=CHCH2)4(CH2)2COOH −50 Animal membranes
of double bonds. The position of the double bond, starting from the carboxyl group, is indi-
cated by the delta symbol, with the positions shown as superscript. As shown in Table 3.1, the
melting points of unsaturated fatty acids are lower than the corresponding saturated fatty
acid with the same number of carbons. Thus, the presence of double bonds shifts the fatty
acid structure into a more fluid state.
The physical properties of unsaturated fatty acids are the result of the stereochemistry
of the molecule. Two arrangements of alkenes are presented in Figure 3.2: a cis conforma-
tion having both hydrogens on the same side of the molecule and a trans conformation with
attached hydrogens on opposite sides of the molecule. Unlike single bonds, carbon atoms
connected by double bonds cannot rotate, so unsaturated fatty acids are less flexible than
saturated fatty acids. Two unsaturated fatty acids are displayed in Figure 3.3. The trans fatty
acid can assume a straight-chain form, similar to a saturated fatty acid. However, the cis fatty
acid cannot; there is a kink in the chain. As a result, trans fatty acids can pack together as
well as saturated fatty acids. This orderly crystal-like arrangement is analogous to the pack-
ing of shipping boxes. By contrast, cis fatty acids cannot stack together well, accounting for
FIGURE 3.2 Stereochemistry of the double bond. The two possibilities for the stereochemis-
try of double bonds are cis, in which both hydrogens are on the same side of the chain, and trans,
where the hydrogens are on opposite sides.
Chapter 3 – Lipids 37
FIGURE 3.3 Cis and trans fatty acids. The trans fatty acid has a similar orientation to the satu-
rated fatty acids, allowing the molecules to pack in regular arrays. However, the cis oriented
double bond produces a kink in the chain so that packing is interrupted. This distinction causes
the molecules to stack less readily and leads to lower melting points.
FIGURE 3.4 Isolated and conjugated double bonds. Polyunsaturated fatty acids contain more
than one double bond, and they are always isolated, as in the left panel. By contrast, a conju-
gated double bond is shown in the right panel, in which every other carbon is involved in a
double bond, and there is an overlap of the electrons in the π orbitals.
their increased fluidity. Natural fatty acids are all in the cis conformation and have lower
melting points than the corresponding trans fatty acids, a trend illustrated in Table 3.2.
Fatty acids with more than one double bond are polyunsaturated. In naturally occur- TABLE 3.2 Stereochemistry
ring polyunsaturated fatty acids, the double bonds are separated by two single bonds, as
and Melting Points of
illustrated in Figure 3.4. The p–orbitals (see Appendix A2: Chemical Fundamentals) are
drawn for the isolated double bonds of the polyunsaturated fatty acids and compared to Fatty Acids
molecules in which the double bonds are conjugated (the double bonds are separated by Common
one single bond). Isolated double bonds are also called localized, and the electrons of the π Identity Name MP °C
orbitals are relatively more reactive than in conjugated double bonds. Electrons in conjugated 18:0 Stearic 70
double bonds are delocalized as they can be distributed over several orbitals. Delocalization 18:1∆9 - trans Elaidic 44
produces a greater stability (i.e., less reactivity). The conjugation is known as resonance, 18:1∆9 - cis Oleic 16
as illustrated in Figure 3.5. Several pictures of this phenomena are presented, including
38 3.2 Fatty Acids
FIGURE 3.5 Resonance in butadiene. The representations of butadiene show first the mol-
ecule with hydrogens explicitly represented, then with just the carbon backbone. Next, the
electron clouds for the π bonds show the overlap that enables electrons in these orbitals to
delocalize (spread out), which is also known as resonance. The last two representations express
resonance in a carbon framework. The two structures across a double arrow have no indepen-
dent existence; they are different pictures of a single structure to incorporate its properties.
Alternatively, the last representation indicates each carbon can be viewed as having a “bond and
a half” representation.
FIGURE 3.6 Carboxyl group dissociation and resonance. Once the carboxyl group dissociates
to the anion, it has a resonance form, represented by the double arrow method as well as by the
“bond-and-a-half” method. Unlike butadiene, both resonance forms of the double arrow are the
same, which indicates greater resonance stability.
the common double-headed arrow representation of separate structures. It is important to

emphasize that the resonance arrow is not an equilibrium; there is just one chemical species.
The actual structure is a hybrid of multiple forms drawn to represent distinct views of elec-
tron distribution. For the present purposes, we focus on the fact that a resonance structure in
a molecule lends greater stability, thus decreasing reactivity, compared to a similar structure
without resonance.
The yellow color of many oils results from the reactivity of isolated double bonds with
oxygen and light in the fatty acid chains. Within cells, antioxidant systems act to minimize
the same reactions of isolated double bonds in their lipids.
The carboxylic acid head group of the fatty acid (Figure 3.6) has a weak acid’s usual dis-
sociation properties. Its pKa is 4.8, so it is mostly in the anionic form (i.e., —COO−) at pH 7,
which explains why this portion of the molecule is so soluble in water. Once the fatty acid
dissociates, the resulting carboxylate anion is resonance stabilized as indicated. Resonance
stabilization increases product formation (the anion form), accounting for the low pKa of
carboxylic acids. For comparison, a fatty alcohol (R–OH) can only weakly dissociate to a
fatty alkoxide (R–O−) with a pKa of 16.
3.3 TRIACYLGLYCEROLS
Most of the fatty acids found in mammals are in the form of triacylglycerols, which serve
as the major energy depot for the body. The triacylglycerol molecule consists of three fatty
acids esterified to a glycerol backbone as illustrated in Figure 3.7. The ester bond is a linkage
of an alcohol and an acid, releasing water. While the carboxyl group of fatty acids make them
amphiphilic, the ester bond of triacylglycerols does not participate in hydrogen bonding,
making these molecules hydrophobic. Hydrophobicity is an advantage for storage because
excluding water reduces the overall weight of adipocytes (fat cells), essentially vessels con-
taining a large fat droplet each. Many other cell types, such as liver and muscle, also contain
triacylglycerols, but within multiple, smaller vesicles known as lipid droplets. An interme-
diate form is found in another specialized tissue, the brown adipose. Cells of this tissue are
also packed with triacylglycerol, but it is distributed into many small vesicles rather than the
single one in the white adipose (the major form, usually referred to as simply adipose tissue).
FIGURE 3.7 Esters and the triester of triacylglycerol. Ester formation is formally the removal of
water between an alcohol and an acid, as shown in the top portion. This bond occurs three times
between glycerol and three fatty acid molecules to form triacylglycerol.
40 3.3 Triacylglycerols
The brown color results from numerous mitochondria; we will return to brown adipose cells
in Chapter 11.
Whereas animals primarily use lipids (as triacylglycerol) for energy, plants mostly use car-
bohydrates instead. Carbohydrates, having substantial amounts of oxygen, are heavier than
lipids. Weight is not a problem for plants as they are immobile (literally planted). The seed
is the only mobile phase of a plant’s life cycle. Accordingly, lipid storage in plants is mostly
confined to the seed stage. All of our familiar plant lipids are derived from seeds, such as corn
oil, peanut oil, or the generic vegetable oil (which is mostly soybean oil).
Because of the different fatty acids found in triacylglycerols, there is considerable struc-
tural diversity among these molecules. These differences reflect the features we examined in
the prior section: chain length and degree of unsaturation. Saturated fatty acyl chains tend
to form a solid phase, typical of triacylglycerol deposits near organs; these are known as
visceral fat (Box 3.1). Less saturated chains are found in the deposits just beneath the skin,
called subcutaneous fat. Even more unsaturated triacylglycerides are found in the seed lip-
ids; these are referred to as oils. The variation in fatty acyl chains of triacylglycerols can be
appreciated by examining those found in pizza (Box 3.2). Generally, lipids are oils if they are
liquid at room temperature and fats if they are solid at room temperature. The distinction is
somewhat arbitrary; at 0°C (the temperature of STP, or standard temperature and pressure),
most oils would be solids.
Box 3.1 On Steak and Fish: Polyunsaturated Fats, Trans Fats, and Health Risks
A high-quality (prime) steak is said to be “marbled”, which means that it has a large
amount of fat dispersed throughout the meat. When cooked, this mixes with the meat,
solubilizing its flavors, rendering a taste that most omnivores like. Animal fat is more
saturated than fish lipids. Diets rich in animal fat are associated with an increased risk of
cardiovascular disease, for reasons that remain elusive. However, it is known that fatty
deposits in arteries are a prime cause of cardiovascular disease.
Omega fatty acids found in fish appear to promote cardiovascular health. These fatty
acids are polyunsaturated and may act by substituting for some eicosanoids, a group
of lipid-signaling molecules. The “omega” name refers to a numbering system distinct
from the nomenclature used in Table 3.1. For example, linoleic acid has a double bond 6
carbons away from the terminal CH3 group, so it would be called an “omega-6” fatty acid.
Trans fatty acids, because of their structural similarity to saturated fatty acids, would pose
a similar risk but are worse as their metabolism is slower. Trans fatty acids are not found
in nature but are produced synthetically. For example, margarine is made by the chemi-
cal reduction of a mixture of triglycerides (with a reductant such as sodium borohydride).
The reduction of the double bonds is followed by chemical oxidation to introduce a con-
trolled amount of unsaturation, allowing any desired physical consistency, such as “tub
margarine”. Unfortunately, this produces a mixture of cis- and trans- isomers.
Box 3.2 Lipid Composition of Pizza
The lipids found in pizza are almost entirely triacylglycerols, which vary in their fatty
acid composition. These are shown in Figure B3.2. The more abundant fatty acids (those
representing at least 1% of the total) are displayed in Figure B3.2a. Only four fatty acids
account for the majority of types: myristic acid (14:0), palmitic acid (16:0), stearic acid (18:0),
oleic acid (18:1 or, more specifically, 18Δ9), and a mixture of 18:2 fatty acids, including the
common linoleic acid (18Δ9,12). About half of the fatty acids are unsaturated and half are
saturated. Moreover, virtually none of the fatty acids has an odd number of carbons. The
reason for this is that they are synthesized two carbon atoms at a time.
A fuller display of fatty acids shows the wide array of less abundant fatty acids
(Figure B3.2b), which includes those with three and four double bonds. In particular,
FIGURE B3.2 Pie chart of pizza lipids. (a) Major fatty acids. (b) Fatty acid composition.
arachidonic acid (20Δ5,8,11,14), while present in small amounts, is important as a precursor
to a large number of signal lipid molecules called the eicosanoids. There are two listings
for 18:3, which denote distinct positions of the double bonds. One is characterized as n-3
in the omega numbering system (see Box 3.1).
3.4 PHOSPHOLIPIDS
The most common polar lipids (or amphiphiles, having both polar and nonpolar affinities
in the same molecule) are the phospholipids, the principal components of cell membranes.
Phospholipids are derivatives of phosphatidic acid (Figure 3.8). They resemble triacylglycer-
ols, except that the third position of the glycerol is esterified to phosphate.
An isolated phosphate group is called inorganic phosphate. Like carboxylic acids, phos-
phoric acid is a weak acid and can combine with an alcohol to form an ester. Unlike carboxylic
acids, phosphates are polyprotic acids, with multiple equilibria (Figure 3.9). Of the three
pKa values, only pK2 (6.6) is close to physiological pH; the others are near 2 and 12. Inorganic
phosphate is a significant contributor to intracellular buffering; cytosolic pH is about 7.1. In
42 3.5 Cholesterol
FIGURE 3.8 Phosphatidic acid. This molecule is the simplest phospholipid; the third position
(called sn-3) is esterified to a phosphate group. The sn refers to a naming system, an abbreviation
for “stereochemical numbering”.
FIGURE 3.9 Inorganic phosphate: acid behavior. Inorganic phosphate (Pi) has three dissocia-
tions and, thus, three separate dissociation equilibria. Each has a separate pKa: pK1 = 2; pK2 = 6.6;
pK3 = 12.
addition to multiple dissociation equilibria, phosphate can form multiple esters (Figure 3.10).
In both acid dissociation and ester formation functions, the phosphate group behaves as if the
P=O portion and each of the –OH portions can act separately as an acid (Figures 3.9 and 3.10).
In the commonly occurring phospholipid phosphatidylcholine (Figure 3.11a and Box 3.3),
the phosphate group is part of a diester, in which one ester is formed with glycerol and the
other ester with the alcohol choline. Phospholipids have considerable heterogeneity due to
variations in the attached fatty acids at the first two positions of the glycerol backbone. There
are also different alcohols esterified to the phosphate; two examples of such phospholipids
are shown in Figure 3.11b. Membranes also contain less frequently occurring but physiologi-
cally important amphiphiles related to phospholipids, such as amide-containing sphingolip-
ids (see Chapter 14).
3.5 CHOLESTEROL
Cholesterol is the best-known example of steroids, a class of lipids having fused, flat rings.
While popularly viewed as a dangerous component when present in high concentrations in
blood, it is an essential component of animal cell membranes.
FIGURE 3.10 Inorganic phosphate: ester behavior. Inorganic phosphate (Pi) can form the
three esters indicated: a monoester, a diester, or a triester. Biologically, the most common are the
monoester and diester forms.
FIGURE 3.11 Phospholipid molecules. a) The most abundant phospholipid in mammalian membranes is phosphatidylcho-
line. It is a diester, formally joining phosphate and two alcohols: one from the third position of glycerol, the other from choline. b)
Two other common phospholipids of membranes are: phosphatidylethanolamine and phosphatidylinositol.
44 3.6 Lipid–Water Interactions of Amphipathic Molecules
Box 3.3 Lecithin and Emulsification
Biochemists use lecithin (Greek for “egg yolk”) as a synonym for phosphatidylcholine. In
the food and pharmaceutical industries, a mixture of phospholipids extracted from ani-
mals or plants – mainly containing phosphatidylcholine – is known as lecithin. Two com-
mon materials are “egg yolk lecithin” (a mildly redundant term considering the origins) and
“soybean lecithin”. While phosphatidylcholine is the major component of these materials,
they contain other phospholipids (e.g., phosphatidylethanolamine) as well as lysophos-
pholipids (phospholipids with just one fatty acid ester). The preparations are essentially
organic solvent extracts of the biological material and are often used for emulsification.
Emulsification refers to the dispersion of insoluble lipids, such as triglycerides, into
water solutions. Lecithins are commonly used for this purpose, for example, in drug deliv-
ery. The hydrophobic portion of the phospholipid can interact with the triglyceride and
the hydrophilic portion can interact with the solution, spreading the oil throughout the
material. Emulsification is the principle behind the creation of mayonnaise and the for-
mation of dispersed lipids in the first portion of the intestine in the digestive process,
thus allowing the breakdown of dietary lipids.
Cholesterol is a polar lipid, with a preponderance of a hydrophobic region consisting of

four rings attached to a short hydrocarbon chain. The hydrophilic portion is just a hydroxyl
group (Figure 3.12). Many other steroid molecules are derivatives of cholesterol, such as the
sex steroids testosterone and estradiol, and the salt-retention hormone aldosterone, also
shown in Figure 3.12. Cholesterol and steroid derivatives, fatty acids, and phosphatidylcholine
are examples of amphipathic molecules, having both hydrophilic and hydrophobic portions
within the same molecule. We next explore how amphipathic molecules interact with water.
3.6 LIPID–WATER INTERACTIONS OF AMPHIPATHIC MOLECULES
Amphipathic molecules must satisfy two conflicting natures simultaneously: water solubility
and lipid solubility. Two aggregate forms that achieve this balance are micelles and lipo-
somes (Figure 3.13).
In micelles, the polar portions face the aqueous exterior, and the nonpolar portion forms
a core excluded from water. Fatty acids are an example of a molecule that forms micelles in
FIGURE 3.12 Cholesterol and other steroids. Steroids have four flat, fused rings and various
substituents. Both cholesterol and estradiol are alcohols (sterols); testosterone and aldosterone
are ketones.
FIGURE 3.13 Micelles and liposomes. a) The micelle structure allows the polar portion of the
amphiphile to interact with water on the outside of the structure while packing the nonpolar
tails together in the interior. b) In the liposome, two water phases are separated by a nonpolar
interior phase, forming a sphere. A portion of that sphere can be viewed as a bilayer, an impor-
tant conceptual division when considering transport and membrane studies.
water (Figure 3.14). As fatty acids are added to a water solution, their concentration increases
to a point that represents the individual molecule’s solubility in water, called the critical
micelle concentration (CMC). Adding fatty acids at higher concentrations than the CMC
drives the formation of increased numbers of micelles, but the fatty acid concentration of the
solution itself is only slightly increased. Table 3.3 lists CMC values for two fatty acids and
cholesterol. Those with a high CMC, like octanoate, are more soluble in water. Palmitate,
with a very low CMC, is nearly insoluble in water. Cholesterol has an intermediate CMC and
an intermediate solubility in water. The number of carbons (27 in cholesterol, 16 in palmitate)
is not the sole determinant of solubility or CMC unless we compare strictly straight-chain
fatty acids. Unlike other physical constants such as the melting point, the CMC is strongly
46 3.6 Lipid–Water Interactions of Amphipathic Molecules
TABLE 3.3 Critical

Micelle Values of Fats
Molecule CMC (mM)
Palmitate (16:0) 0.002
Octanoate (9:0) 400
Cholesterol 0.040
FIGURE 3.14 Critical micelle concentration. Micelle forming molecules, such as the fatty acids,
have a break in their concentration-solubility curves as they coalesce to form micelle aggregate.
The concentration at which fatty acids form micelles is called the critical micelle concentration,
and the dramatic increase occurs as shown.
dependent on experimental conditions, such as salt concentrations. As a result, the number

of molecules in each micelle can vary. Thus, while useful, CMC is only an empirical measure
of the molecular aggregate.
An alternative to micelle formation is the liposome (Figure. 3.13). This aggregate con-
sists of three phases: an exterior water phase, an interior water phase, and a nonpolar phase
sandwiched between them. For example, phosphatidylcholine forms liposomes in water, and
the resulting structure is the essence of a biological membrane. Figure 3.13b also shows an
expansion of a portion of the liposome structure, indicating that an alternative description
of this structure is the bilayer.
The two different types of aggregates resolve the conflicting solubility demands in dis-
tinct ways. To approach the problem of why a lipid structure would form one aggregate or
the other, consider the relative volumes of the hydrophobic and hydrophilic portions of the
molecules (Figure 3.15). If these are unequal, then the molecules tend to pack like slices of a
pie (albeit in three dimensions) and form a micelle. If they are roughly equal, they will pack
FIGURE 3.15 Rationale for micelle or liposome/bilayer formation. Micelles form when the
amphiphiles have different volumes for the polar head and nonpolar tail structure and pack like
wedges to create a sphere. When the volumes occupied by each are approximately equal, the
individual molecules can stack like bricks and form bilayers, which can wrap to form liposomes.
like bricks, forming a bilayer that, when wrapped around itself to form a sphere, becomes a
liposome. While this notion of “packing” may seem simplistic, it is similar to the formation
of crystals. For example, fatty acids have an unequal lipid and polar portion, indicating that
they pack as wedges and form micelles. Other micelle-forming lipids may not have the same
packing-unit shape but have hydrophilic and hydrophobic regions with unequal volumes.
The hydrophilic and hydrophobic regions in phosphatidylcholine have roughly equal vol-
umes, so this amphiphile packs as a bilayer, and forms a liposome in water. Physiologically,
we are usually most interested in bilayers since this is the form of cellular membranes. One
biological example of micelles is the formation of particles containing bile salts in the small
intestine lumen. These particles also include cholesterol and some phospholipid and serve to
absorb dietary lipids, facilitating their transfer and hydrolysis to fatty acids, which can then
enter the epithelial cells lining the small intestine.
3.7 PHOSPHOLIPID MONOLAYERS
A distinct barrier function of lipid molecules is the formation of a monolayer of phospholipid

molecules. It is possible to experimentally construct a monolayer by layering phospholipids
on a water surface – essentially an oil slick (Figure 3.16a). This arrangement of phospholipids
allows the phospholipid tails to interact with air, which is also hydrophobic, and the head
group of the phospholipid to interact with water. A lipid species that assumes this structure
is also known as a surfactant.
One biological example of a monolayer is found in the air–water interface of lung epithelia
as illustrated in Figure 3.16b. These epithelial cells form small air-facing sacs (numbering in
the hundreds of millions) known as alveoli, the lung functional unit. The exchange of gasses
(CO2 and O2) occurs across the membrane of the alveolar epithelial cells. Maintaining the
alveoli as separate structures, thereby creating a large surface area for gas exchange, is accom-
plished by an exterior monolayer of dipalmitoyl phosphatidylcholine. Air is hydrophobic, and
the external cell membrane surface is hydrophilic. The monolayer prevents the coalescence of
the alveoli, which otherwise would be driven by the surface tension of water (a consequence of
the hydrophobic effect introduced in the last chapter). The significance of the lung monolayer
is evident in the pathology of respiratory distress syndrome, in which newborns do not
form adequate amounts of secreted phosphatidylcholine at birth and the air sacs do not form.
The other biological form of monolayers is illustrated in Figure 3.16c. In this case, the
head groups of the phospholipids face the aqueous exterior and the tails point to the inte-
rior. While similar to the micelle, in this case, the interior contains distinct hydrophobic
molecules, most of which are triacylglycerols and fatty acid esters of cholesterol such as
palmitoyl-cholesterol:
H3C
CH3
H3C
CH3
CH3
O O
Palmitoyl-cholesterol ester
48 3.7 Phospholipid Monolayers
(a) Air (hydrophobic)
Water (hydrophilic)
(b) Air
Epithelial cell
Water
(c) Lipid interior
(d) (e)
Plasma membrane Plasma membrane
Lipid droplets Lipid droplets
Cytosol Cytosol
(f)
Surface monolayer
Lipid interior
FIGURE 3.16 Monolayers. Monolayers are a single two-dimensional aggregation of amphi-

philes. The hydrophilic portion of the lipid interacts with a water phase, and the lipid tail inter-
acts with a hydrophobic phase. (a) a monolayer separates air–water phases in vitro. (b) In lung
alveoli (air sacs), the hydrophilic portion of phosphatidylcholine contacts the lining cells (epi-
thelia), and the hydrophobic fatty acyl groups interact with the air (hydrophobic phase). (c) In a
lipid droplet, the monolayer is the “skin” that separates the inner lipid phase from the cytosol, an
external water phase. The lipid phase consists mainly of triacylglycerols and cholesterol esters.
(d) Multiple lipid droplets form in many cell types. (e) The adipocyte has just one lipid droplet,
but it occupies most of the cell volume apart from a small cytosol between the monolayer and
the plasma membrane. (f) A lipoprotein particle has a structure similar to a lipid droplet. These
particles are found extracellularly in the blood plasma and are responsible for exchanging lipids
between cells.
The lipid interior is, therefore, a storage depot of triglycerides and cholesterol esters. In
most cells, these structures exist as multiple small lipid droplets, as shown in Figure 3.16d.
An extreme case is the white adipocytes (or just fat cells), in which there is only one lipid
droplet that takes up most of the cell volume (Figure 3.16e). A thin cytosol contains all cell
inclusions such as mitochondria, nucleus, and endoplasmic reticulum. The cytosol forms a
narrow rim between the plasma membrane and the monolayer of the lipid droplet. Finally,
there are lipid droplets that are exterior to cells and exist instead within the bloodstream.
These are known as lipoprotein particles. As shown in Figure 3.16f, their lipid structure is
very much like a cellular lipid droplet, except that lipoprotein particles serve the function of
lipid exchange between different tissues connected by the blood.
3.8 LIPID COMPOSITION OF MEMBRANES
The plasma membrane and internal cell membranes are bilayers (apart from the monolayers
of lipid droplets). The phospholipid molecules of biological membranes show considerable
structural variation. Part of this variation is in the phospholipid head group, which may be
other alcohols in place of choline (see Figure 3.11). Another factor contributing to phospho-
lipid diversity is the different fatty acyl chains, which lead to changes in membrane flu-
idity. This concept is analogous to solid–liquid phase transitions in other molecules but,
in membranes, refers to the relative ability of the individual molecules to move within the
bilayer. The presence of double bonds in the fatty acyl chains favors an increase in fluidity.
Polyunsaturated lipids have a further increase in fluidity, although the extent of this increase
above that of one double bond is known to be modest.
The presence of cholesterol also contributes to the fluidity of membranes. At high tem-
peratures, cholesterol tends to decrease the fluidity of the membrane; at low tempera-
tures, it increases it. Thus cholesterol, which is most abundant in outer leaflets of plasma
membranes of animal cells, tends to dampen temperature-induced changes in membrane
fluidity. There is more to the biological function of cholesterol than its effect on fluidity,
as this molecule is essential for mammalian cell growth. There is structure specificity to
this action, as cholesterol cannot be replaced with structurally similar sterols. Cholesterol
interacts with membrane proteins, which are themselves essential for cell function. In
yeast, ergosterol rather than cholesterol is present in membranes and required for the
growth of this organism.
CH 3
H 3C CH 3
CH 3
CH 3
H 3C
HO
Ergosterol
3.9 WATER PERMEABILITY OF MEMBRANES AND OSMOSIS

The liposome serves as a model for cellular membranes. One consequence of a mem-
brane separating two water phases is that each water phase can contain molecules at
50 3.9 Water Permeability of Membranes and Osmosis
different concentrations. Unlike most hydrophilic substances, water itself can readily
cross the membrane. This transport occurs despite the limited solubility of water in the
membrane core, due to the immense concentration of water. Additionally, the flow of
water is assisted by the membrane fluidity imparted by the unsaturated fatty acyl chains
in most membranes. In some cellular membranes, water flow can be further accelerated
and regulated by the presence of aquaporins, which are protein channels selective for
water transport.
Most of the channels and transport proteins in cell membranes exist to transport polar
solutes (such as Na+ or glucose) across the membrane. Selective transport is another way
of saying that physiological bilayers are semipermeable. The process can be modeled by a
classic experiment using a U-tube apparatus illustrated in Figure 3.17. A glass tube is bent
into a U shape, and a semi-permeable membrane is placed in its center. Small dots represent
permeable water molecules, and large dots represent an impermeable solute dissolved in
water.
The first frame (Figure 3.17a) shows two solutions the instant after they were placed on
separate sides of the membrane. The left side is a solution, and the right side has pure water.
Water can then flow unimpeded from right to left, as indicated in Figure 3.17b. This flow will
increase the left side’s volume and thus its height; this flow is known as osmosis. The driving
force is diffusion: this is the movement of molecules from a region of high to low concentra-
tion, a statistical tendency (see Box 3.4). As water moves to the left side, it dilutes the solute
there. Simultaneously, the rising fluid on the left side creates a pressure that forces water back
somewhat. Figure 3.17b is, therefore, a balance of these forces.
In Figure 3.17c, a piston has been introduced on the left side, and pressure added to equal-
ize the fluid levels on both sides of the U tube. The gauge indicates the amount of pressure
needed to create this condition; its value is known as the osmotic pressure of the solution.
The water introduced into the right side is free of solute. This procedure is also the principle
behind reverse osmosis, a method to desalinate water.
In this chapter, we have examined structural features of the major lipid species: tri-
acylglycerols, phospholipids, and sterols. There are a variety of other lipids that have
specialized biological purposes. Many polar lipids are conjugates with other types of
molecules, such as lipopolysaccharides, which are lipids covalently bound to carbohy-
drates. To understand these molecules, we next examine the structural properties of
carbohydrates.
FIGURE 3.17 Osmosis in the U-tube. a) Solute (large dots) is dissolved in one solution, and
pure water (small dots only) is present in the other. Initially, these are placed in the U-tube at
equal volumes. b) Due to diffusion, water moves from right to left, which also tends to equalize
its concentration across the semi-permeable membrane. In (c), a piston is introduced into the left
side to push the solution down so that both sides become equal again. The pressure reading on
the gauge (pressure = force × area) indicates the solution’s osmotic pressure.
Box 3.4 Word Origins: Osmosis
Osmosis originated as a technical term to describe water movement in cells. Originally

separated as “endoosmosis” and “exoosmosis”, the simplification to osmosis reflects the
Greek origins of the word, meaning a push or impulse. It is a simple driving force based
on an even simpler one: diffusion. Molecules move by diffusion because there are more
of them in one place than another; statistically, their locations tend to even out. When
this occurs across a membrane, and when this membrane is permeable to water but not
to other molecules, the phenomenon of osmosis emerges, as described in the text. Long
associated with water movements within plant cells and across the membrane of all cells,
the non-scientific use of the word suggests that the process is mysterious: the ability to
obtain new knowledge by its flow into the brain from the nether regions.
SUMMARY
Lipids are used primarily for energy storage and membrane formation. Their dominant prop-
erty is insolubility in water. The building block of most lipids is the fatty acid, which consists
of a hydrophobic hydrocarbon tail and a carboxylic acid head. When the tail contains double
bonds, the fatty acid is said to be unsaturated. The double bond in naturally occurring fatty
acids is in the cis configuration, with both hydrogens on the same side of the chain. The more
unsaturated and the shorter the chain, the more soluble the molecule is in water. When the
carboxylic group head groups of fatty acids form ester bonds with the alcohols of glycerol,
the result is a triacylglycerol. These are fully hydrophobic molecules and serve as the primary
energy storage form for the body. Phospholipids are similar to triacylglycerols, except that
the third alcohol of glycerol is esterified to phosphate. In the common phospholipid molecule
phosphatidylcholine, the phosphate is also esterified to the alcohol choline; this is therefore a
diester. Both phospholipids and the steroid molecule cholesterol are found in cell membranes,
and both are amphiphiles, having both hydrophilic and hydrophobic portions in the same
molecule. The fatty acids are also amphiphiles. Micelles are formed when the volumes of the
two portions of amphiphiles are different, such as in fatty acids. Micelles have a hydrophobic
core and hydrophilic exterior. Liposomes form when the hydrophobic and hydrophilic por-
tions of the amphiphile have similar volumes, as in phosphatidylcholine. A distinct form of
barrier is the monolayer, in which a two-dimensional association of phospholipids separates
a hydrophilic from a hydrophobic environment. An example is the surface of the air-filled
lumen of the lung. Monolayers also comprise the outer surface of lipid droplets (intracellular
lipid storage vesicles) and the outer surface of the lipoproteins (blood-borne particles that
exchange lipids between tissues of the body). When a liposome or a membrane forms, water
can pass through the bilayer, but most molecules cannot; the barrier is semi-permeable. Such
barriers permit a flow of water to balance the concentrations on both sides, called osmosis.
REVIEW QUESTIONS
1. Bacteria that flourish in extremes of temperature have several chemical strategies

that allow them to adapt. What sort of fatty acyl chains would you expect to find in
extreme thermophiles (those living near the boiling point of water)?
2. If the pKa of an average fatty acid is about 5, what effect would there be on solubility
at pH values of 2, 5, and 7?
3. What features make a phospholipid suitable for a membrane but not an energy stor-
age molecule? What about the features of triacylglycerols that make it suitable for
energy storage but not membrane function?
4. Commercially available oils from plant seeds are typically yellow, but fats are typi-
cally almost colorless. Explain the reason for this difference.
52 Chapter 3 Addendum: Inverted Micelles
5. In the U-tube apparatus, suppose the solute could also pass through the membrane,
but more slowly than water. What would be the result of this setup over time?
6. Red blood cells are commonly used to illustrate the dramatic actions of osmosis. A
solution that is lower in osmotic strength than the red cell cytosol is called “hypo-
tonic”; one that is higher is osmotic strength is called “hypertonic”, and one that is the
same is called “isotonic”. Explain what each type of solution would do to a red cell.
7. Soap is made by hydrolysis of triglycerides, using lye (NaOH). The resulting solution
contains sodium ions, fatty acids at alkaline pH, and glycerol. The fatty acids form a
micelle when in solution. Explain how the micelle permits the action of soap in wash-
ing your hands.
8. Detergents also contain micelle-forming fatty acids used in cleaning but are consid-
ered more potent. Rather than having a carboxyl group, they have a sulfate or phos-
phate group. Suggest why this might make them more effective in cleaning.
9. Most sterols are more hydrophilic than cholesterol. Very hydrophilic sterols include
the bile salts, which have several hydroxyl groups added to the ring, and usually a
water-soluble modification and shortening of the chain attached to the five-mem-
bered ring of the sterol. This modification includes an acid function, such as a carbox-
ylate or sulfate. Why are bile salts sometimes called bile acids? Which form of the bile
acid exists at pH 8, which prevails in the intestine?
10. Why can’t fatty acids themselves be used as the storage form of lipids?
11. An unusual type of fatty acid found in some bacteria is a cyclic fatty acid such as lac-
tobacillic acid:
OH
If this were part of a membrane phospholipid, how would it affect the fluidity com-
pared to saturated fatty acids?
12. Most of the fatty acids in the lung surface monolayer are long chain saturated fatty
acids (C16:0). What do you think is the reason for this?
CHAPTER 3 ADDENDUM: INVERTED MICELLES
We have examined the common types of amphipathic lipid aggregates: micelles, lipo-
somes, and monolayers. Another form of lipid aggregate is an inverted micelle, in which
the water-soluble portion forms an interior space, and the hydrophobic portion is exterior
as illustrated in Figure 3CA.1a. As symbolized in the figure, a pure lipid that forms this
structure has a cone shape; the unequal volumes of hydrophilic and hydrophobic portions
FIGURE 3CA.1 Inverted micelles. (a) Pure inverted micelles of diacylglycerol. (b) An inverted
micelle inside a membrane (c) Membrane fission and fusion involve inverted micelles.
suggest a micelle structure. However, in this case, the hydrophilic portion is small and
directed inward, and the hydrophobic portion is on the exterior. Several lipids can form
this structure, including anionic lipids such as phosphatidylserine or phosphatidylinositol.
Those lipids are part of membranes and, thus, cannot dominate the physical properties of
the membrane, so they must be considered a minor part of the total phospholipid pool.
Another molecule that forms this structure is diacylglycerol. This molecule is not part
of bilayers but can be formed from the chemical splitting of a phospholipid to release the
phosphate and the base portion.
When diacylglycerol is formed in a membrane, it can assume an interior portion that sat-
isfies the isolation of the hydrophilic and hydrophobic portions as shown in Figure 3CA.1b.
One of the consequences of this structure is to anchor the hydrophilic portions of proteins
within the membrane. A separate function is illustrated in Figure 3CA.1c: membrane fis-
sion and fusion. The progression shown indicates how transient inverted micelles are formed
from membrane phospholipids and ultimately form separate vesicles.
(Source: J. Jouhet, Importance of the hexagonal lipid phase in biological membrane orga-
nization, Front Plant Sci, 4 (2013) 1–5.)
KEY TERMS
amphiphiles
aquaporins
bile salts
brown adipose
critical micelle concentration (CMC)
diester
diffusion
eicosanoids
emulsification
fatty acids
inorganic phosphate
isolated double bond
lecithin
lipid droplet
lipids
lipoprotein
liposomes
membrane fluidity
micelles
nonpolar lipids
oils
osmosis
osmotic pressure
polar lipids
polyprotic acids
polyunsaturated
resonance
saturated
semipermeable
stereochemistry
steroids
sterols
subcutaneous fat
surfactant
unsaturated
visceral fat
54 Bibliography
BIBLIOGRAPHY
Doyle E. Trans fatty acids. J. Chem. Educ. 74: 1030–1032, 1997.
Nawar WW. The article contains more detail on the chemistry of formation and the health conse-
quences of the trans-fatty acids. The author concludes with the intriguing observation that a
Pseudomonas bacteria has an enzyme that enables conversion of cis to trans fatty acyl chain
in the membrane to decrease membrane fluidity when the organism enters a stationary phase.
Chemical changes in lipids produced by thermal processing. J. Chem. Educ. 61: 299–302, 1984.
Segel IH. The author describes changes in lipids induced by heat, with comments on light alteration of
the fatty acid components. Biochemical Calculations: How to Solve Mathematical Problems in
General Biochemistry. New York: Wiley, 1976, p. 441.
Stewart PA. Among other subjects, this book contains numerous exercises in acid dissociations. How
to Understand Acid-Base. A Quantitative Primer for Biology and Medicine. New York: Elsevier,
1981, p. 186.
Tanford C. This is an eclectic view of acid-base in whole-body physiology. The level is simple but
underscores the importance of viewing all equations in dissociation behavior in a system. The
approach can be applied to simple solutions up to the human body. The Hydrophobic Effect:
Formation of Micelles and Biological Membranes. New York: Wiley, 1980.
Jain MK. This is a good source for the general properties of lipids and their ability to form different
phases, as well as data for the interaction between lipids and water. Introduction to Biological
Membranes. New York: Wiley, 1988, p. 423.
Cantor CR and Schimmel PR. This book has a detailed description of membranes, including the dis-
tinction between bilayer and micelle formation. Many details of different membranes and dif-
ferent possible phases of lipid materials are also present. Biophysical Chemistry. San Francisco:
W. H. Freeman, 1980.
Van Holde KE, Johnson WC, and Ho PS. This is a detailed description of physical biochemistry,
including a good explanation of both diffusion and osmotic pressure. Principles of Physical
Biochemistry. Upper Saddle River, NJ: Pearson/Prentice Hall, 2005, p. 710, [27].
Yeagle, P., This is a more concise physical biochemistry text, with appropriately more brief treatments
of diffusion and osmosis. The Structure of Biological Membranes, 3rd ed. Boca Raton, FL: CRC
Press, 2012.
Carbohydrates 4
Carbohydrates are most commonly encountered as the sweetener sucrose and as starch, the
nutrient of bread and pasta. The carbohydrates are literally hydrates of carbon, having the
empirical formula (CH2O)n. Because they possess both hydroxyl and carbonyl groups, they
are a step up in complexity from lipids. Unlike lipids, carbohydrates are generally very soluble
in water. The Latin word for sugar is saccharide; this is commonly used in the term polysac-
charides. Sugars are nutrients for both animals and plants. Animals use sugars for rapid (i.e.,
minute-to-minute) energy production and lipids for long-term storage. Plants use sugars for
energy exclusively, except at the seed stage. Sugars have greater weight than lipids, making
them less suitable for long-term energy storage for organisms with mobile lifestyles (i.e., ani-
mals and seeds). The reason for the greater mass of sugars is two-fold. Sugars contain more
oxygen atoms and are thus denser than lipids. Sugars also associate with water (through
hydrogen bonding), further increasing their weight.
Aside from providing energy, sugars have a wide variety of roles once they are chemically
modified, such as cell–cell recognition and signaling. The specificity of blood group types
derives from sugars attached to membrane lipids of red blood cells. Proteins secreted from
cells are usually covalently linked to sugars. Thus, sugars are integral to biological function.
We begin our study of sugars with the simplest ones: the monosaccharides.
4.1 MONOSACCHARIDES
Monosaccharides have a single carbonyl group, which occurs at the first or second carbon
atom. All remaining carbons have an attached hydroxyl group. There are two classes of
monosaccharides: aldoses and ketoses. Aldoses (aldehydes) have the carbonyl at carbon one,
whereas ketoses (ketones) have the carbonyl at carbon two.
The “-ose” suffix (as in aldose and ketose) usually indicates that a compound is a sugar. The
general category name for sugars is shown in Table 4.1, starting with the smallest, the triose.
Exceptions to this naming rule occur in the simplest monosaccharides: dihydroxyacetone
and glyceraldehyde.
OH O
H2 C HC
C O CH OH
H2 C H2 C
OH OH
Dihydroxyacetone Glyceraldehyde
Dihydroxyacetone is a symmetrical molecule because a plane of symmetry can be drawn

through its center. However, glyceraldehyde is asymmetric: there are two distinct arrange-
ments of the four groups attached to its central carbon. These forms are illustrated in Figure 4.1
using a Fischer projection to represent three-dimensional molecules in a plane. The two-
dimensional Fischer projection convention is that the horizontal attachments project above
55
56 4.1 Monosaccharides
TABLE 4.1 General

Nomenclature of Sugars
Carbons Category Name
3 Triose
4 Tetrose
5 Pentose
6 Hexose
7 Heptose
8 Octose
FIGURE 4.1 d-and l-glyceraldehyde. These mirror images are shown as Fischer projections
and three-dimensional views. Asterisks indicate chiral carbons.
d
C
FIGURE 4.2 Tetrahedral carbon. The four groups attached to the central carbon are as far
apart from one another as possible, forming a tetrahedron in space.
the plane, and the vertical attachments project behind the plane. A further requirement is
that the first carbon – the most oxidized – is shown on top. Another representation of the
central carbon and its attached groups is a virtual tetrahedron, as shown in Figure 4.2.
The two forms of glyceraldehyde are called stereoisomers, and molecules that display
this form of stereoisomerism are known as enantiomers. The central carbon to which the
four distinct groups are attached is the chiral carbon; any molecule containing such a car-
bon is known as a chiral molecule. A naming system for chiral molecules is based on glyc-
eraldehyde. The form with the hydroxyl group attached to the chiral carbon on the left side
is called l (for Levo); the other enantiomer is called d (for Dextro). These names are Latin for
“left” and “right”; the word “chiral” itself is Latin for “hand”. Just as your left and right hands
are not superimposable on each other, the two enantiomers cannot be rearranged in a way
that causes them to be superimposed; they are distinct molecules.
Chapter 4 – Carbohydrates 57
Enantiomers share many chemical properties, such as identical melting and boiling points;
these are among the very features used to establish the unique identity of a molecule! An
experimental demonstration of the distinction between enantiomers is their interaction with
polarized light. Polarized light is produced by passing light through special filters that elimi-
nate all light ray directions other than those oriented in one plane. Experimentally, the inter-
action of a compound with polarized light is measured as degrees of rotation from a reference
point, and a separate naming system is used (Box 4.1). From a biochemical perspective, the
Box 4.1 Stereochemical Conventions: Big d/l, Little d/l
In addition to the d/l naming convention, a separate system uses the lower-case letters d
and l (alternatively, + and −) to refer to the direction of rotation of plane-polarized light:
d or + for clockwise and l or − for counterclockwise. To better understand the use of the
d/l system, consider the following practical experiment. Suppose you take a pair of polar-
ized sunglasses, pop out the lenses, and place them at either end of a tube filled with
water (Figure B4.1). This device is a polarimeter. If you look through the tube, rotating
one sunglass lens to a particular point will completely prevent the passage of light. If you
continue to rotate it, the light will appear again until, with continued rotation, it darkens
once again. Mark the darkest point. This is where the polarized planes in the lenses are
at right angles to one another. This follows from the operation of a Polaroid filter, which
allows light beams to travel only in one direction. If you now replace the water in the tube
with a solution of an optically active (chiral) molecule and start with the marked point,
one lens will have to be rotated to some extent to restore the dark point. That displace-
ment is measured in degrees, right or left (i.e., d or l). Rotation of the plane of polarized
light reveals the presence of chiral molecules in the solution.
FIGURE B4.1 Polarimetry. A pair of polarized sunglasses can be used as polarizing filters.
Removing the lenses and placing each on opposite sides of a sealed tube filled with a solu-
tion to be analyzed is the essence of a polarimeter. (a) The tube is filled with water. Both of
the filters are oriented in the same way, allowing the passage of maximal light. The polariza-
tion is indicated by lines on the filters, showing that they are in parallel. (b) The filters are now
oriented at 90° to one another, which prevents light from passing. (c) The tube is filled with a
d -glucose solution. To restore maximum darkness, one filter is rotated clockwise. The result-
ing rotation value indicates that glucose is a d-sugar or, equivalently, a + sugar. Because this is
independent of absolute stereochemistry (d/l status), the name includes both designations:
d -(+)-glucose. The same experiment performed with d -fructose would show that the filter
would require rotation in the counterclockwise direction; hence, fructose is d-(–)-fructose.
58 4.1 Monosaccharides
FIGURE 4.3 d-erythrose. This is a d sugar as the configuration of the chiral carbon at position
3 – the one furthest from the carbonyl – aligns with d-Glyceraldehyde. The other chiral carbon
at position 2 is defined by naming the resulting structure “erythrose”. Asterisks indicate chiral
carbons.
greatest significance of enantiomers is their selective interaction with enzymes. For example,
most living systems can metabolize d-sugars but not l-sugars.
Larger sugar molecules (e.g., those with four or more carbon atoms) have multiple chiral
carbons. Figure 4.3 shows the Fischer projection of d-erythrose, a four-carbon sugar. It is an
aldose because the first carbon is an aldehyde group. The next two carbons are both chiral,
as indicated by asterisks. The naming convention for sugars with multiple chiral centers is
to start with the assignment of the d or l form. The rule is that the carbon furthest from
the carbonyl determines if the entire molecule is d or l. In this example, it is carbon 3. By
comparing this molecule to the reference sugar glyceraldehyde, this erythrose is a d-sugar.
All that remains is to resolve the ambiguity of the remaining chiral center. This is done by
simply assigning a name to the molecule: erythrose. Our focus will be on d-sugars because
they are far more common than l-sugars. The other four-carbon d-sugars are d-threose and
d-erythrulose.
HC H2C OH
HO C H C O
H C OH H C OH
CH2OH CH2OH
D-Threose D-Erythrulose
Unless we are making an explicit stereochemical point, we will usually omit the “d” indicator.
Sugars that differ at chiral centers other than the d and l positions are called epimers.
Thus, threose is an epimer of erythrose. Larger sugars have correspondingly greater numbers
of isomers, but the naming conventions hold. After assigning the d and l forms, each molec-
ular arrangement of the remaining chiral centers has a unique name. Examples of commonly
occurring d-sugars are ribose, glucose, galactose, and fructose.
O O
CH CH H2C OH
O
HC HC OH HC OH C O
HC OH HO CH HO CH HO CH
HC OH HC OH HO CH HC OH
HC OH HC OH HC OH HC OH
CH2OH CH2OH CH2OH CH2OH

Ribose Glucose Galactose Fructose
4.2 RING FORMATION IN SUGARS
Sugars having five or more carbons exist in solution primarily in a cyclic form. The reaction
that leads to ring formation in sugars is an intramolecular version of the more general one
that forms hemiacetals or hemiketals (Figure 4.4). As described in the figure, the reaction
joins a carbonyl group with a hydroxyl group (a nucleophilic addition). Note that the carbonyl
oxygen becomes a hydroxyl group in the product.
The carbonyl carbon in the straight chain form of the sugar becomes the anomeric car-
bon in the ring form. Because both the carbonyl and alcohol groups come from the same
molecule, the reaction forming a hemiacetal or hemiketal is more favorable than if the reac-
tants were from different molecules. The other consequence of this intramolecular reaction
is that the product becomes a ring. The most stable rings have five or six members. In sugar
solutions, they comprise about 99% of the forms and are typically the only ones illustrated.
Open-chain and ring forms for the sugars fructose and glucose are shown in Figure 4.5.
The representation of ring forms as joined, straight-chain diagrams in Figure 4.5 is drawn
to emphasize the relationship between the open-chain and ring structures. However, as a
chemical representation, the bond lengths and angles are unrealistic. A more accurate rep-
resentation is the Haworth Projection, shown in the middle structure of Figure 4.6. When
constructing this diagram, you need only place the groups appearing on the right side of the
FIGURE 4.4 Hemiacetal and hemiketal formation. The reaction of an alcohol group with a
carbonyl is one of simple nucleophilic substitution.
60 4.2 Ring Formation in Sugars
FIGURE 4.5 Ring formation in sugars. The reaction shown in Figure 4.4 takes place intramo-
lecularly in sugars having at least five carbons. Because the carbonyl and alcohol are part of the
same molecule, the product is a ring.
FIGURE 4.6 Views of sugar rings. The straight-chain form (Fischer projection) can be recast
as the Haworth projection by taking groups positioned to the right in the Fischer projection
above the ring. The chair form is a solution structure that more closely represents the three-
dimensional structure of the sugar molecule.
FIGURE 4.7 Multiple forms of glucose. The two ring forms of glucose are each in equilibrium
with the open-chain form.
straight-chain Fischer projection below the ring in the Haworth projection to achieve the
correct orientation. Note that the Haworth is a simplified representation of the more realistic
chair form, also shown in Figure 4.6. The chair form allows attachments of each carbon to
assume a tetrahedron.
We now turn our attention to the hydroxyl group of the anomeric carbon. In the linear form
of a sugar, this is a carbonyl. However, in the ring form, it is an alcohol and a new chiral center.
As expected with a chiral carbon, there are two stereochemical forms of the molecule, each in
equilibrium with the straight-chain form (Figure 4.7). We need a designation for this new form
of stereochemistry. Consider the position of the hydroxyl group at the anomeric carbon (for
glucose, carbon 1). If this hydroxyl group is on the opposite side of the ring from the carbon
substitution determining the d-form of the sugar, it is the alpha form. The top ring form sugar
of Figure 4.7 is therefore designated formally as α-d-glucose. In the other ring form, the two
groups – the connections to C1 and C5 – are on the same side; this is the beta form. As a quick
check, with the ring oriented as in Figure 4.7, the newly appearing hydroxyl group is below the
ring in the α-anomer and above the ring in the β-anomer. Notice that each form is in equilib-
rium with the open-chain form so that it is possible to convert the α form to the β form by going
through the open-chain intermediate. At equilibrium, less than one percent of all the glucose in
solution is in the open-chain form. The distribution of ring forms is about 36% alpha and 64%
beta. This preference stems in part from the axial position of the –OH group in the alpha form
and its equatorial position in the beta form. Equatorial substitutions on six-membered rings
have less steric hindrance and are thus more stable than axial substitutions. A more complete
explanation for the preference for beta substitutions is presented in Box 4.2.
To summarize, we have discussed three types of stereochemistry that allow us to unam-
biguously describe a sugar:
1. d or l, which refers to the configuration of the chiral carbon furthest from the car-
bonyl group.
2. Different names for other epimers (e.g., mannose or galactose).
3. Alpha or beta, which refers to the configuration of the anomeric carbon.
The ring forms of ribose and fructose, shown in Figure 4.8, are also in equilibrium with their
open-chain forms. The fact that common sugars form rings is important not merely because
this is the predominant solution form, but also because the hydroxyl group attached to the
anomeric carbon is used in bonding sugars together. The first example of this kind of linkage
is found in the disaccharides.
62 4.3 Disaccharides
Box 4.2 The Anomeric Effect
The ring forms of common sugars predominate in solution, as discussed in the text. There
are substantial amounts of alpha and beta forms (about 36% and 63%, respectively) of
glucose. Still, this distribution cannot be fully explained by steric preference for the equa-
torial position of the beta form. Focusing on the structure of the anomeric carbon, the
structures are:
OH
OH
α-D-glucose β-D-glucose
and it is clear that the equatorially positioned beta form should be relatively free of ste-
ric hindrance from other ring substituents. However, by making comparisons to similar
molecules, including the cyclohexane ring, there is substantially more alpha form than
would be expected from steric forces alone. This unexpected preference was observed
in the 1950s and called the anomeric effect. It is a stabilizing interaction between the
orbitals of the bridge oxygen and the C–OH bond of α-anomeric carbon. The details of
this interaction remain controversial, but mechanism aside, it is clear that the alpha form
is thereby favored more than would be expected on steric considerations alone.
FIGURE 4.8 Ring forms of ribose and fructose.
4.3 DISACCHARIDES
Two sugars bound together are called disaccharides; some examples are shown in
Figure 4.9. The best known is sucrose; it is what we popularly call “sugar” itself. Sucrose is
ubiquitous in plants as the energy substrate is transferred between cells. Sucrose is heav-
ily concentrated in just a few organisms, such as sugar cane and sugar beets. Maltose is a
product of the partial breakdown of starch and an intermediate in the production of beer.
The partial digest itself is called a malt, hence the name malt liquor. Trehalose is com-
monly found in bacteria, fungi, and invertebrates and plays a role analogous to sucrose in
those organisms. Lactose is the milk sugar, which is produced by mammals for feeding
their newborns.
The bond between the two sugars in disaccharides is known as a glycosidic bond. A
glycosidic bond links the hydroxyl group of an anomeric carbon of one sugar to a hydroxyl
group of a second sugar. This bond is diagrammed in Figure 4.10 for the case maltose.
The actual metabolic route of formation is more elaborate, requiring several intermedi-
ate steps. Maltose is formally designated glucose-(α1→4)-glucose. This nomenclature
specifies that the first sugar uses its anomeric carbon (the C1 of glucose) in the alpha
orientation, and forms a covalent bond to the C4 hydroxyl group of the second glucose.
Once the glycosidic bond is formed, the anomeric carbon in maltose is locked into the
alpha form. However, the glucose attached to it on the right side can still establish an equi-
librium between the open chain and the beta forms. Thus, there are three separate forms of
maltose; once again, the ring forms predominate.
Disaccharides – or any sugar polymer – in our diet must be hydrolyzed to monosac-
charides prior to entering the cells lining the small intestine. The reaction is catalyzed
by enzymes positioned extracellularly and specific to each digestible disaccharide: i.e.,
sucrase is required to hydrolyze the glycosidic bond of sucrose, and lactase is required
to hydrolyze that of lactose. In some cases, this process is impaired, leading to digestive
disorders (Box 4.3).
In the disaccharides of Figure 4.9, one of the sugar units is glucose; the other is glucose,
galactose, or fructose. When just one anomeric carbon is involved in glycoside formation, as
in maltose and lactose, a free anomeric carbon remains in equilibrium with an open-chain
form (see Figure 4.10 for maltose). Even a small amount of this open-chain form in solu-
tion allows the carbonyl group to cause the reduction of metal ions in diagnostic tests. The
positive result in this experimental test indicates the presence of a reducing sugar. Thus, all
monosaccharides are reducing sugars; of the disaccharides displayed in Figure 4.9, maltose
and lactose are reducing sugars, while sucrose and trehalose are nonreducing sugars. An
exploration of the term reducing is presented in Box 4.4.
FIGURE 4.9 Disaccharides. A glycosidic bond links the two sugars in a disaccharide. This bond
locks the anomeric hydroxyl in either the alpha or the beta configuration. Either one (maltose
and lactose) or both (sucrose and trehalose) sugar units have their anomeric carbons engaged
in a glycosidic bond.
64 4.4 Polysaccharides
FIGURE 4.10 Maltose as a reducing sugar. Maltose is a disaccharide of one glucose in the
alpha form covalently linked to the 4-hydroxyl of a second glucose. This second glucose mol-
ecule is in equilibrium with an open chain form as well as the beta ring form. The presence of the
carbonyl in the open chain form of the second glucose moiety makes maltose a reducing sugar.
4.4 POLYSACCHARIDES
When more than two sugars are linked via glycosidic bonds, they are said to be either oli-
gosaccharides (meaning “a few”) or polysaccharides (meaning “many”). The distinction is
inexact: some consider chains of up to ten linked sugars to be oligosaccharides. Commonly,
polysaccharides have thousands of monosaccharide residues bound together. Such molecules
have extremely large molecular weights and have new properties not found in smaller mol-
ecules. In general, large molecules that are constructed from small units are called poly-
mers. The “building block” units may be the same or slightly different from one another.
The polysaccharides are our first example of biological polymers; the other two major classes
Box 4.3 Medical Connections: Sugars and Digestion
Sugars are normally absorbed as a monosaccharide by the small intestine. If appreciable

amounts are not removed by the time they reach the large intestine, they are metabo-
lized there by the resident bacteria. The bacterial end products include gases that cause
distension of the colon, bloating, and pain. Two of the most common causes of this bloat-
ing are lactose intolerance and raffinose. Lactose intolerance is a common genetic defi-
ciency of lactase, an enzyme that catalyzes the hydrolysis of lactose. Lactase is present
in abundance in infants and less so in adults. Because lactose is only present in milk, and
milk is a normal component of the diet only in infancy in all animals but humans, lactose
intolerance is strictly a human problem.
A common intestinal disturbance is caused by raffinose, a sugar found in foods such as
cabbage and beans. Raffinose is poorly digested due to the presence of the galactose–
(α1→6)–glucose bond in the first two sugar moieties of the trisaccharide. As a result, it is
metabolized in the large intestine, with attendant gas production. However, due to the
far smaller amounts of raffinose in those foods, the clinical symptoms of raffinose diges-
tion are less severe than those of lactose intolerance.
Box 4.4 Word Origins: Reducing
When a word with such rich meanings as reducing is applied to sugar chemistry, its inter-
pretation may be obscured. There are different sociological, mathematical, biological,
and chemical definitions for this word. In the everyday sense, the word originally meant
to bring back or restore an original condition. A later use was related to conquest or sub-
ject to a social lowering.
Mathematically, the term refers to the factor-label method of manipulating units alge-
braically in a separate way from the numbers they label, expressed as “reducing to lowest
terms”. In biology, meiosis involves a reductive division, in which the number of chromo-
somes is halved. In chemistry, reduction is the decrease in oxidation number for an atom.
A compound is reduced if it gains electrons, so the term reduction is somewhat mis-
leading as a description of the chemical process. In the context of this chapter, a reduc-
ing sugar transfers electrons from the carbonyl group to cause reduction of a metal. For
example, an aldehyde such as glucose can reduce a blue solution of Cu2+ in the presence
of hydroxide ions to cuprous oxide (Cu2O), a red precipitate. In the product, copper has an
oxidation state of +1, a chemical reduction.
None of these definitions describes the dieter’s dream molecule: a truly reducing
sugar. Still, there is a long history of artificial sweeteners. Sucralose is a modern artificial
sweetener made by chlorinating sucrose:
OH OH
Cl
O O
Cl HO OH
HO HO
O OH OH
HO
O OH
O
O
Cl OH
HO OH
Sucralose Sucrose
As a result, sucralose can attach itself to the taste receptors (the sweet receptors in
the tongue) cells, but is metabolically inert. Thus, sucralose provides the sweetness of
sucrose, but none of the calories.
Sucrose itself is the standard of sweetness for both artificial and natural sugars. For
example, sucralose is 600 times sweeter than sugar. The measurement scale is less
66 4.4 Polysaccharides
objective than others because it requires human tasters. This method is especially trou-
blesome for discovering new sweeteners that, unlike sucralose, may bear no structural
similarity at all to sugars, making it impossible to predict in advance whether a com-
pound is a potential candidate. Typically, a sweetener is discovered by accident; someone
working with a chemical product has tasted it (and survived!).
are proteins and nucleic acids. While lipids can form large aggregates with new properties,
they are not biological polymers because they consist of noncovalently associated small mol-
ecules. Nonbiological polymers, such as nylon, were discovered and studied at the same time
as biological polymers. Nonbiological and biological polymers share some properties, includ-
ing the methods used for their analysis.
4.4.1 LINEAR POLYSACCHARIDES
Amylose is a biological polysaccharide composed of glucose molecules linked via αl→4 gly-
cosidic bonds (Figure 4.11). Like all linear polymers, the amylose chains have distinctive
ends, called the reducing end and the nonreducing end. This property of dissimilar ends
also applies to disaccharides. Maltose also has a reducing end and a nonreducing end, albeit
a chain of just two monosaccharide units. All unbranched polysaccharides have exactly one
reducing end and one nonreducing end. In solution, amylose forms hydrogen bonds between
water molecules and the free hydroxyl groups at positions 2, 3, and 6. These linear chains are
extended polymers with considerable flexibility.
Cellulose, another linear polymer of glucose, has β1→4 linkages and assumes the very regu-
lar three-dimensional solution structure shown in Figure 4.12. The linear strands are arranged
in parallel, with all of the hydroxyl groups engaged in hydrogen bonds. The total intramolecular
hydrogen bonding provides considerable internal strength, and it also renders cellulose unable
to bind to water. This polymeric structure is possible only with the beta-position of the ano-
meric carbons in the glycosidic bonds. Thus, cellulose is entirely insoluble in water, despite the
presence of multiple hydroxyl groups (Box 4.5). Cellulose is the most abundant molecule in the
world, principally found in plant cell walls; it is the major component of wood.
One reason polysaccharides serve as energy storage molecules involves osmosis
(Chapter 2). Osmolarity depends only on the number, rather than the size (or other qualities),
of molecules. A glucose polymer counts as a single molecule no matter how many glucose
subunits it contains. This substantially reduces intracellular osmolarity compared to indi-
vidual glucose molecules, minimizing osmotic pressure while maintaining a virtual glucose
reservoir within the cell. This balance is critical for animal cells, which must maintain the
same osmolarity on either side of the plasma membrane. By contrast, plants simply have a
FIGURE 4.11 Amylose has distinct ends. The straight-chain glucose polymer amylose has
α1→4 bonds. The glucose residue at the left end of the chain has its anomeric carbon engaged
in a glycosidic bond; this is the nonreducing end. Every polysaccharide has nonreducing and
reducing ends. The lower figure shows a simplified representation of a linear polysaccharide,
emphasizing the connections of the unit glucose molecules.
FIGURE 4.12 Cellulose. Cellulose does not interact with water because all of its hydroxyl
groups are engaged in intracellular hydrogen bonding. As a result, cellulose is completely insol-
uble in water.
Box 4.5: THE IRONY OF INTRAMOLECULAR HYDROGEN BONDS
Hydrogen bonding is a key feature that makes certain compounds soluble in water.
However, when the groups responsible for hydrogen bonding are linked to a group other
than water, they lose their solubility. This type of hydrogen bonding occurs when the
linkages are intramolecular. Cellulose, a polymer of glucose found in plants, is an example
of a compound that is insoluble in water due to intramolecular hydrogen bonds. These
kinds of interactions exist in all biological polymers, however, including nucleic acids and
proteins.
A practical application of this property is the development of synthetic molecules
called methylcellulose. These are formed in the laboratory by creating methyl esters
with some of the hydroxyl groups of cellulose. Partial methylation prevents the remain-
ing free hydroxyl groups from forming internal hydrogen bonds, creating new poly-
mers whose partial solubility in water can be controlled. These polymers can be used
as solvents for lipid-soluble drugs. The glucose molecules in cellulose are connected by
beta linkages, for which humans have no enzymes to break down, so methylcellulose
is biologically inert. The irony is that by creating methyl esters and preventing hydro-
gen bond formation on the sugar rings, the molecules become more water-soluble. The
explanation is that creating partial esterification of hydroxyl groups removes intracel-
lular hydrogen bonding, leaving the remaining hydroxyl groups free to hydrogen bond
to water.
cell wall – for which cellulose is a key structural component – allowing plant cells to main-
tain very high internal osmotic pressures without bursting.
68 4.5 Carbohydrate Derivatives
FIGURE 4.13 A polysaccharide branch point. The α1→6 bond, found in glycogen and amylo-
pectin, creates a branch point in the polysaccharide structure. With one branch point, there are
now two nonreducing ends and still one reducing end.
4.4.2 BRANCHED POLYSACCHARIDES
Amylopectin is a branched polysaccharide that occurs in plants. In addition to αl→4 link-

ages, amylopectin also has αl→6 linkages, commonly called branch points. Figure 4.13
shows how a single αl→6 glycosidic bond creates a branch in the overall structure. Branched
polysaccharides have a more compact three-dimensional structure than linear chains.
Starch is a mixture of amylose and amylopectin that occurs in plants. Different plants
have various proportions of the two polysaccharides, with varying polymer lengths and
degrees of branching, accounting for the distinctive qualities of corn starch, potato starch,
and wheat starch. The more ubiquitous wheat flour is mostly starch, along with considerable
amounts of protein from the seed (up to 15%).
The branched polysaccharide glycogen serves as rapidly accessible intracellular energy
storage in animal cells. Structurally, glycogen has the same bonds as amylopectin, except
that glycogen contains even more α1→6 branch points and is larger. While glycogen exists in
various animal cells, its concentration is highest in the liver and muscle. Liver glycogen con-
tent fluctuates with the availability of glucose. The liver stores glycogen in the fed state and
breaks down glycogen to free glucose units in the fasted state. By contrast, muscle takes up
blood glucose and uses glycogen’s glucose residues for its own metabolism during active con-
traction. Thus, the liver’s role in glucose balance is altruistic, providing for the entire body,
whereas the muscle’s role is selfish, being used only for muscle metabolism. Most glycogen
molecules in the human body are found in skeletal muscle, which has about 1% of its weight
as glycogen. The liver can have up to 10% of its weight as glycogen (during the fed state) but
represents less of the total body glycogen due to its relatively small total mass.
4.5 CARBOHYDRATE DERIVATIVES
Many molecules have many of the qualities of carbohydrates because they are metabolically
derived from them. We will consider two categories of such derivatives: simple modifications
and substituted carbohydrates. The simple modifications consist of only slightly modified
monosaccharides and polysaccharides. In the more heavily substituted carbohydrates, the
sugar’s role is limited to its connections; the chemical properties of the original sugar are lost.
4.5.1 SIMPLE MODIFICATIONS
The modification of hydroxyl groups by a phosphate ester occurs in many of the metabolic
intermediates we will encounter in later chapters. For example, glucose-6-P (the phosphate
group is abbreviated as P) is glucose with a single phosphate ester, and fructose-1,6-bis-P2 has
two phosphate esters (Figure 4.14). Another simple modification is a change in the oxidation
state of one of the carbons. Reduction of the carbonyl carbon results in a sugar alcohol, or
polyalcohol, such as glycerol (Figure 4.15). Glycerol is a product of fat breakdown and a major
additive in prepared foods and drugs. Conversely, oxidation of the carbonyl group produces
a sugar acid, such as glyceric acid. Oxidation can also occur at other carbons, leaving the
carbonyl group intact, as in glucuronic acid (Figure 4.16). Note that glucuronic acid can still
form a ring and become part of a polymer, but gluconic acid can do neither.
Replacing an oxygen atom with a nitrogen atom produces another derivative, exemplified
by glucosamine
FIGURE 4.14 Phosphorylated sugars.
FIGURE 4.15 Redox derivatives of sugars.
HO
O
OH
OH
HO
NH2
Glucosamine
which is found as a subunit of secreted proteins and in connective tissue. Derivatives in

which one of the hydroxyl groups is replaced with a hydrogen atom, as in deoxyribose
FIGURE 4.16 Two oxidized glucose derivatives. Oxidizing glucose at the anomeric carbon
forms gluconic acid, which can no longer form a ring or become polymerized. On the other
hand, glucuronic acid, an oxidation product at C6 carbon, can still form a glycosidic bond and
thus be part of a polysaccharide.
HO
O OH
HO
2-Deoxyribose
are called deoxy sugars.

Polysaccharides can also be modified to produce derivatives with altered properties. For
example, the molecule agarose (found in certain seaweeds) has a repeating d-galactosyl unit
linked with a β1→4 glycosidic bond to a modified l+++++-galactose residue. The latter con-
tains an internal bridge oxygen between carbons 3 and 6 and a sulfate group esterified to car-
bon 2 (Figure 4.17). Agarose is a linear molecule of this repeating unit, although not all of its
l-galactosyl groups are sulfated. A similar polysaccharide is agaropectin, which has branch
points. Together, the mixture of agarose and agaropectin is known as agar.
4.5.2 SUBSTITUTED CARBOHYDRATES
Nucleotides, lipopolysaccharides, and proteoglycans all contain sugar molecules, but they
are so heavily modified by other groups that their chemical features greatly diverge from less
substituted sugars.
Nucleosides are modified sugar molecules attached to a nitrogenous base in an
N-glycosidic link, as in the example of adenosine:
FIGURE 4.17 Agarose. The repeating unit of the polysaccharide agarose, a linear modified
polysaccharide. The two residues shown are a galactosyl unit and a modified galactose in a
β1→4 linkage.
FIGURE 4.18 Purines and pyrimidines.
NH2
N
N
N N
HO O
OH OH
Adenosine
The base moieties are either pyrimidines or purines, as shown in Figure 4.18. The sugar
moiety is either ribose or deoxyribose, as shown in the examples of Figure 4.19. Note that the
nitrogen attached to the sugar is the 1-position of the pyrimidines but the 9-position of the
FIGURE 4.19 Nucleosides. A base from those shown in Figure 4.18 is attached to a ribose
sugar or a deoxyribose sugar. Not every combination is possible: uracil is not found attached to
deoxyribose, and thymine is not found attached to ribose.
FIGURE 4.20 ATP, ADP, and AMP. Nucleotides are phosphorylated nucleosides. The three
examples shown here are the most common nucleotide forms of adenine used in energy trans-
fer reactions.
purines. The sugar portion is further substituted in nucleotides: a phosphate is esterified to

one of the available hydroxyl groups. These can be monophosphates or chains of phosphates.
In phosphate chains, a diester bond forms between two phosphate moieties. Figure 4.20
shows the three common nucleotides known as AMP, ADP, and ATP.
Nucleotides are key energy transfer molecules for metabolic reactions in cells. Additionally,
nucleotides are joined together via phosphodiester bonds to form the polymers DNA and
RNA. The sugar component of DNA is deoxyribose, and the sugar component of RNA is
ribose. DNA exists as two strands noncovalently linked by hydrogen bonds between the
bases (Figure 4.21). The sugar component of DNA (i.e., deoxyribose) has no remaining free
hydroxyl groups. A hydrogen atom substitutes the hydroxyl group at C2; the C1 is engaged in
an N-acetyl bond to the base; the other two form phosphate diester linkages that comprise
the covalent backbone of each strand. RNA, unlike DNA, is usually a single-stranded poly-
mer (Figure 4.22).
DNA provides a template for two fundamental cellular processes. First, the molecule can
duplicate itself in preparation for cell division, an activity known as replication. Second,
FIGURE 4.21 DNA. The two strands of the double-stranded DNA molecule are linked by
hydrogen bonding. Within each strand, the sugars are covalently linked via phosphate diester
bonds. The “ribbon” structure shows that the entire molecule forms a helix, an example of a
polymeric higher-order structure.
small regions of DNA can serve as a template for messenger RNA (mRNA) formation, an
activity known as transcription. In turn, mRNA provides a template for protein synthesis
(translation). These processes are critical to the creation of new cells or new proteins for
cells.
Sugars form conjugates with other biological molecules. For example, some phospho-
lipids are sugar-lipid derivatives such as phosphatidylglycerol and phosphatidylinositol
(Figure 4.23). Sugar-protein conjugates (called glycoproteins) are usually divided into two
classes: O-linked and N-linked (Figure 4.24a and b). In the O-linked example, a glycosidic
bond is formed between the sugar (the modified sugar N-acetylgalactosamine is shown) and
a hydroxyl group from a protein, here contributed from the side group of an amino acid
called serine. The N-linked example in Figure 4.24b shows an N-glycosidic bond, similar to
those found in nucleotides. Here, the nitrogen is contributed by the side group of an amino
acid called asparagine. An additional protein-sugar conjugate arises when an amine from a
lysine side chain in hemoglobin reacts with glucose, followed by rearrangement to the stable
FIGURE 4.22 RNA. Like DNA, the sugar portion of RNA binds the chain together, using phos-
phate diester bonds, as shown. The purine and pyrimidine bases also are attached to the sugars
of RNA, like those of DNA. Unlike DNA, the RNA molecule is typically a single chain.
FIGURE 4.23 Phosphatidylglycerol and phosphatidylinositol.
ketone shown in Figure 4.24c. This relatively slow reaction produces a form of hemoglobin
abbreviated as HbA1c. Measuring HbA1c levels provides a measure of the average blood
glucose over several weeks. HbA1c, therefore, is of considerable importance in monitoring
diabetics.
FIGURE 4.24 Sugar-modified proteins. The most common modifications are (a) O-linked
and (b) N-linked polysaccharides; these are glycosidic and N-glycosidic bonds, respectively. (c) A
nonenzymatic reaction of glucose with hemoglobin produces a specially modified glycosylated
hemoglobin.
SUMMARY
Sugars have an empirical formula of (CH2O)n, with multiple hydroxyl groups and one car-
bonyl group in the simple sugars. The smallest monosaccharides are the trioses glyceralde-
hyde and dihydroxyacetone. There are two stereochemical forms of glyceraldehyde because
it has a chiral center. These are denoted d and l, but biological sugars are overwhelmingly in
the d form. Sugars containing more carbon atoms have correspondingly larger numbers of
stereoisomers, which are identified by assigning unique names. For example, one six-carbon
sugar is glucose; another, differing in configuration at the 4-carbon, is galactose, the 4-epi-
mer of glucose. The common five- and six-carbon sugars form rings in a reaction between a
carbonyl group and a hydroxyl group. The formation of rings produces a new chiral center
because a new hydroxyl group emerges from the carbonyl group. The carbon of this group is
the anomeric carbon and is always involved in bonds linking sugars together. Multiple sugars
may be covalently bonded as disaccharides, like sucrose and maltose, or polysaccharides,
such as glycogen and cellulose. The link between sugars is called a glycosidic bond, and it
locks the position of the anomeric hydroxyl into two separate stereochemical forms (α and
β). The three-dimensional structure that results from this distinctive orientation is the dif-
ference between glycogen, the water-soluble storage carbohydrate in animals, and cellulose,
the water-insoluble structural carbohydrate of plants. Any sugar that contains a free ano-
meric carbon (hemiacetal or hemiketal) can equilibrate with an open-chain form. Because
it can react with a test solution and reduce an indicator ion, it is called a reducing sugar. All
monosaccharides and polysaccharides, and some disaccharides such as maltose, are reduc-
ing sugars. Sucrose is a nonreducing sugar because both of the anomeric carbons of glucose
and fructose are part of the glycosidic bond. Polysaccharides have at least one nonreduc-
ing end – more if there are branches. A myriad of carbohydrate derivatives exist in biol-
ogy, both of the simple monosaccharides (such as phosphorylated sugars and deoxy sugars)
and the polysaccharides (such as the sulfated polymer agarose). More elaborate substitutions
produce molecules where the properties of the sugar itself are lost, and the sugar merely
becomes a connecting molecule, such as the role deoxyribose plays in the structure of DNA.
76 Review Questions
Finally, sugars form conjugates with other biological molecules, such as lipopolysaccharides
and proteoglycans.
REVIEW QUESTIONS
1. Raffinose is a sugar that, upon hydrolysis of its glycosidic bonds, yields galactose,
glucose, and fructose. The galactose–glucose bond is an α1→6 linkage, and the trisac-
charide is a nonreducing sugar. Draw the structure of raffinose.
2. In the DNA structure, the sugar is completely modified. List each modification and
identify an analogous, simpler substitution that exists in another sugar molecule.
What sugar properties remain in the DNA molecule?
3. Glucose in solution has two ring forms and one open-chain form. How many struc-
tures exist in equilibrium for a polysaccharide of glucose with multiple α1→4 bonds
and three α1→6 bonds?
4. Reducing sugars are a mixture of alpha and beta forms. Why does one predominate
over another in different sugars?
5. Related to the previous question, suppose you have equal glucose and fructose con-
centrations at room temperature. In both cases, beta-ring forms predominate, but
with different percentages. However, in each case, when a reducing sugar measure-
ment is made, the same amount of indicator ion is reduced. Moreover, the amount
of directly reacting species for the indicator ion is far less than 1% of the total forms.
Explain these findings.
6. One indication of the distinction in physical properties between simple sugars (like
monosaccharides and disaccharides) and polysaccharides is their observed behavior
in solution. For example, only starch can form a paste in water. Speculate on how that
can be explained from the structural differences between these types of molecules.
7. An early test for sugars is the chemical oxidation of glucose using CuSO4 in the
presence of a base. This copper salt is blue; when heated with sugar, it produces the
reduced form of copper, CuO, a red precipitate. It was used to measure the elevated
glucose in diabetics and in the original discovery of insulin. Why isn’t it used today?
8. How are sugar polymers and lipid micelles similar? What are the distinctions between
these structures?
CHAPTER 4 ADDENDUM: THE DISCOVERY OF STEREOISOMERISM
In the mid-19th century, Louis Pasteur was engaged in the study of the salts of tartaric acid.
O OH
OH
HO
OH O
Tartaric Acid
While the structure was not known at the time, it was a common substance, as it one of the
major acids present in grapes and retained in wine, responsible for much of its flavor. White
crusts of tartar are common on wine casks. A similar compound had been isolated prior to
Pasteur’s work, which had virtually identical properties except that it was not optically active;
that is, it did not cause rotation of polarized light. The polarimeter preceded the discovery
of optical isomerism by several decades. The optically inactive other compound was actu-
ally a mixture of d and l-tartrates. It was named “paratartaric acid”, but was also known as
FIGURE 4CA.1 Tartrate crystals.
FIGURE 4CA.2 Stereoisomers of tartaric acid.
racemic acid, from which we derive the name racemic mixture for any pair of stereoiso-
meric molecules.
Pasteur made crystals of paratartare solutions and discovered two distinct crystal shapes
(using a hand magnifying glass) (Figure 4CA.1). Notice the crystal shapes have notches
(inclined facets) on opposing faces and that the left and right-handed crystals are not super-
imposable on each other. Imagine rotating the right crystal about the long axis; the facet
doesn’t match up with the left crystal.
Next, Pasteur made a solution of separated left and right crystals. While the original
solution of paratartrate showed no optical rotation, the solutions of separated crystals each
showed a definite rotation: the same value but opposite signs. Thus the paratartrate was
resolved into l-tartrate and d-tartrate; this terminology refers to the sign of rotation in the
polarimeter (see Box 4.1).
Later studies based on these observations with tartrates led to the notion of a chiral car-
bon center and the tetrahedral carbon itself, even though the example that started it all is
itself more complex. There are two chiral centers in tartaric acid, which increases the num-
ber of distinct forms. As illustrated in Figure 4CA.2, the enantiomeric forms themselves are
not due to reflection about a chiral carbon, but the center of the molecule. There is a third
form, another stereoisomer, but not an enantiomer, the meso form, which only occurs when
molecules contain multiple chiral carbons. Here, the molecule has a central asymmetry
plane. Thus, there is just one meso form, yet it is a distinct third stereoisomer of tartaric acid.
Tartrate crystals can be visualized even today as precipitates from wine. They may appear
around the cork, particularly if the wine is stored at lower temperatures. Wine purveyors
insist they are completely harmless; to assure oenophiles, they have named these crystals
“wine diamonds.”
KEY TERMS
agar
agaropectin
agarose
78 Bibliography
alpha (form)
anomeric carbon
anomeric effect
branch points
cellulose
chiral
disaccharides
enantiomers
epimers
Fischer projection
glycogen
glycosidic bond
Haworth projection
hemiacetals
hemiketals
ketoses
lactose intolerance
methylcellulose
nonreducing end
nonreducing sugars
nucleotides
oligosaccharides
polymers
polysaccharides
polarimeter
raffinose
racemic mixture
reducing end
reducing sugar
replication
saccharide
sugar acid
transcription
translation
triose
BIBLIOGRAPHY
H. Bunn, K. Gabbay, P. Gallop. The Glycosylation of Hemoglobin: Relevance to Diabetes Mellitus.
Science 200 (1978) 21–27.
J.M. DeMan. Discovery of glycosylated hemoglobin. Principles of Food Chemistry. A Chapman & Hall
Food Science Book. Aspen Publishers, Gaithersburg, MD. (1999) 163–208.
D.E. Drayer. A treatise on the chemistry of food; the chapter on carbohydrates includes many of the
biochemical properties of sugars in detail. The Early History of Stereochemistry: From the
Discovery of Molecular Asymmetry and the First Resolu90tion of a Racemate by Pasteur to the
Asymmetrical Chiral Carbon of Van't Hoff and Le Bel. Clinical Research & Regulatory Affairs
18 (2001) 181–203.
M.J. Paul, L.F. Primavesi, D. Jhurreea, Y. Zhang. Trehalose Metabolism and Signaling. The central role
of Louis Pasteur is recounted in this review, in context of several other scientific giants. Annual
Review of Plant Biology 59 (2008) 417–441.
S. Ugidos-Rodriguez, M.C. Matallana-Gonzalez, M.C. Sanchez-Mata. While trehalose is not an energy
molecule in most plants, it is part of a regulatory system in plants. This review provides some
background to trehalose in general, and to trehalose and details trehalose as a plant signal mol-
ecule. Lactose Malabsorption and Intolerance: A Review. Food & Function 9 (2018) 4056–4068.
Incidence, description, and treatment of lactose intolerance.
Amino Acids and Proteins 5
The word protein is of Greek origin, meaning “first place” or primary. Berzelius originated
this term in 1838 to identify a substance found in plant fibers essential for animal nutrition.
This identification was well before the molecular nature of proteins was discovered. Proteins
are the most diverse of biomolecules. They include structural proteins (such as the plant
fibers), binding proteins (such as hemoglobin), and enzymes (such as sucrase). In this chapter,
we examine protein structure and some elements of their binding behavior. In the next, we
examine their function as enzymes. The proteins also play a central role in all subsequent
chapters of the book, commensurate with their paramount importance in biochemistry.
5.1 COMMON STRUCTURE OF THE AMINO ACIDS
All amino acids contain an amine group (most commonly a primary amine) and a carboxyl
group (the acid portion) attached to the same carbon. The latter is called the α-carbon, from
an organic chemistry nomenclature system that assigns Greek letters to carbons adjacent
to carboxyl groups: α, β, etc (Figure 5.1). Note that this is a distinctive use of the Greek let-
tering system from the carbohydrates. Also bound to the α-carbon is a hydrogen atom (the
α-hydrogen) and a variable group designated as R (Figure 5.2). Twenty different R groups
make up the common amino acids, meaning those incorporated into proteins. Except for
glycine (for which R is a hydrogen atom), the α-carbon is chiral and designated as l or d by
comparison to the reference molecule glyceraldehyde, illustrated in Figure 5.3 for the case of
alanine (in which R is –CH3). The carboxyl group of alanine is most similar to the carbonyl
group of glyceraldehyde. The amine group of alanine is most similar to the OH group of glyc-
eraldehyde. In nature, virtually all amino acids are present in the l form.
5.2 BIOLOGY OF THE AMINO ACIDS

The twenty amino acids are presented in Table 5.1, showing their original biological sources
and the year of their discovery. Most amino acids have been known since the 19th century.
For some amino acids, a brief description of their significance is highlighted. For example,
glutamine has the highest concentration in any amino acid blood and serves as a nitrogen
carrier between tissues. That role can sometimes be taken over by asparagine in times of
stress, particularly in some plants. Plants containing high concentrations of asparagine can
also be a source of the toxin acrylamide during cooking.
NH2
acrylamide
Some amino acids, like glycine, are directly used as a neurotransmitter. Glutamate binds a
taste receptor located on the tongue known as the umami receptor. This discovery created
79
80 5.3 Amino Acid Individuality: The R Groups
FIGURE 5.1 Organic carboxyl bearing chain numbering. This system is usually used only for
the first few carbons adjacent to a carboxyl group.
FIGURE 5.2 General amino acid.
FIGURE 5.3 Amino acid stereochemistry. Glyceraldehyde is the reference compound; most
biological amino acids have the L-configuration.
a new category of taste receptors beyond sweet, salt, sour, and bitter. The sodium salt of
glutamate is marketed as a taste additive to provide a meaty taste to foods, known as MSG
(monosodium glutamate).
We begin our study of the amino acids and their roles in proteins by examining their
chemical structures and properties.
5.3 AMINO ACID INDIVIDUALITY: THE R GROUPS
The R-groups chemically distinguish amino acids from one another. Table 5.2 lists the names,
abbreviations, and pK values for the 20 amino acids commonly found in proteins. We catego-
rize them by their polarity and their chemical functional groups.
5.3.1 POLARITIES
The overall polarity of amino acids depends on their R groups. Figure 5.4 shows the struc-
tures of the polar amino acids. We can further subdivide them into three categories: neutral,
Chapter 5 – Amino Acids and Proteins 81
TABLE 5.1 Origins of the Amino Acids

Year
Amino Acid Origin Discovered Non-protein Significance
Alanine Silk protein 1888
Arginine Lupine (a flowering legume) 1886 Direct precursor to urea in liver Urea Cycle
Asparagine Asparagus 1806 Accumulates in plants under stress
conditions; can be a source of acrylamide
Aspartic acid Asparagine hydrolysis 1827
(marshmallow root)
Cysteine Adhesive protein in the cocoon 1865
of the silk moth
Sericum (L): silk
Glutamate Gliadin (wheat seed protein) 1866 Sodium salt (MSG) is the meaty taste,
stimulating the umami taste receptor
Glutamine Beet; gliadin 1883 Nutrient for rapidly dividing tissues, blood
stream nitrogen carrier
Glycine Gelatine; glycine means “sweet” 1820 Neurotransmitter
Histidine Sturgeon 1896 Histamine precursor
Isoleucine Beet 1904
Leucine Cheese 1819
Lysine Casein 1889
Methionine Casein 1921 One-carbon metabolism
Phenylalanine Lupine 1881
Proline Casein 1901
Serine Silk protein 1865 Neurotransmitter precursor
Threonine Oat protein 1925
Tryptophan Casein 1901 Serotonin precursor
Tyrosine Casein 1946
Valine Animal tissue extracts 1856
acidic, and basic. Figure 5.5 shows the nonpolar amino acids, which are further divided into
alkyl chains, branched-chains, aromatics, and a pair of unique nonpolar structures.
5.3.2 FUNCTIONAL GROUPS
Figure 5.6 presents a chemical categorization of some of the amino acids. Here, acids include
not only aspartate and glutamate, but also cysteine, histidine, and tyrosine, as these can act
as acids under some biological conditions. All of the pK values of the side chains of these
acids are shown in Table 5.2. The bases shown in Figure 5.6 are lysine, arginine, and histidine.
The duplicate categorization of histidine means that it can act as both an acid and a base. To
understand this phenomenon, consider first the side chain alone, which is an imidazole ring:
H H
N N N
H+
(5.1)
H+
N N N
H H
Protonation of the neutral histidine ring at the left side produces a resonance-stabilized
intermediate with electrons spread over both nitrogens of the ring and the carbon atom
between them. Dissociation of this intermediate can proceed either by reversal of the first
equilibrium or by loss of a proton from the other nitrogen to produce the molecule on the
right side of Equation (5.1). Of course, both of these molecules are the same. The situation is
TABLE 5.2 Properties of the Amino Acids

Name Abbreviations pK COOH pK NH3 pK R-Group
Alanine ala A 2.35 9.87
Arginine arg R 2.18 9.09 13.8
Asparagine asn N 2.18 9.09
Aspartic acid asp D 1.88 9.6 3.65
Cysteine cys C 1.71 10.78 8.33
Glutamic acid glu E 2.19 9.67 4.25
Glutamine gln Q 2.17 9.13
Glycine gly G 2.34 9.6
Histidine his H 1.78 8.97 5.97
Isoleucine ile I 2.32 9.76
Leucine leu L 2.36 9.6
Lysine lys K 2.2 8.9 10.28
Methionine met M 2.28 9.21
Phenylalanine phe F 2.58 9.24
Proline pro P 1.99 10.6
Serine ser S 2.21 9.15 16
Threonine thr T 2.15 9.12 16
Tryptophan trp W 2.38 9.39
Tyrosine tyr Y 2.2 9.11 10.07
Valine val V 2.29 9.74
FIGURE 5.4 Polar amino acids. Three subdivisions shown are: acidic, basic, and neutral.
slightly different for the amino acid histidine because the ring is substituted. The ring num-
bering system is indicated in Figure 5.7, and the same equilibria now produce a distinct mol-
ecule of histidine with the hydrogen attached to a N1 instead of N3. The ability of imidazole
or histidine to produce either product from the resonance intermediate explains the low pK
of histidine. A further important feature of histidine is its participation in enzyme catalysis
(Chapter 6). It has been estimated that histidine residues take part in half of all known enzy-
matic mechanisms.
FIGURE 5.5 Nonpolar amino acids. Three major subdivisions are: alkyl, branched-chain, and
aromatic. Methionine and proline are the single examples of a thioether and secondary amine,
respectively.
FIGURE 5.6 Functional groups in amino acids. A cross-categorization of amino acids that
groups together all acids, bases, and hydroxyl groups. Cysteine and methionine are single exam-
ples of the functional groups of sulfhydryl groups and thioether, respectively.
The three amino acids bearing hydroxyl groups in Figure 5.6 are distinct. Serine has a
primary hydroxyl group, threonine has a secondary hydroxyl, and tyrosine has an aromatic
hydroxyl group (a phenol). We can glean some insight from the pK values for these groups
listed in Table 5.2. While serine and threonine each have a pK of 16, tyrosine has a pK of
10. The latter is due to the aromatic ring of tyrosine that stabilizes the deprotonated, nega-
tively charged form (Figure 5.8). By contrast, the deprotonated serine or threonine forms an
unstable alkoxide anion on deprotonation:
NH3
NH3
(5.2) O + H
OH
OOC
OOC
FIGURE 5.7 Imidazole equilibria. Three forms of the imidazole ring – the functional group of
histidine – are shown to be in equilibria. Protonation of either of the neutral forms (tautomers)
produces the same resonance stabilized charged form. Ring numbering is indicated in the first
tautomer.
FIGURE 5.8 Aromaticity of the phenolate ring of tyrosine. Once tyrosine is deprotonated, the
negatively charged phenolate group has several resonance forms, demonstrating aromaticity in
the ring.
The instability of the alkoxide ion product is reflected in the very high pK value. It is also
apparent that the distinction between a secondary and a primary alcohol, at least from the
standpoint of the pK, is slight.
The alkyl and aromatic hydroxyl groups are also distinctive in forming phosphate esters.
The phosphate ester that we observed in the phospholipids of Chapter 3 is an alkyl phos-
phate. As we might expect, this has similar stability as the phosphate esters of serine and
threonine. However, the phosphate ester of tyrosine is much less stable due to the aromatic
stability of the deprotonated tyrosine form.
Sulfur appears in two amino acids: as a sulfhydryl in cysteine and a thioether in methio-
nine. Unlike oxygen, sulfur is a soft nucleophile because its valence electrons are more dis-
tant and more shielded from the positive charge of the nucleus. Nucleophilic addition with S
as the nucleophile is more readily reversed than with O as the nucleophile. In proteins found
in the cell exterior, two separate cysteine –SH groups can form a bridge with each other, the
–S–S–, or disulfide, link. This oxidation reaction occurs in the absence of enzymes. Proteins
inside cells are primarily in the sulfhydryl (–SH) form, maintained by a sulfhydryl-reducing
system in the cytosol.
For all amino acids, alterations in the pH also affect the molecules’ overall charge due
to their dissociable acidic and basic groups. The influence of pH on the overall charge of an
amino acid is the topic we consider next.
5.4 ACID–BASE PROPERTIES AND CHARGE
5.4.1 TITRATION AND NET CHARGE
In Chapter 2, we defined acids as species that raise the [H+] and bases as species that lower
the [H+] (the Arrhenius definition). With this definition, molecules having no hydrogen ion,
such as CO2, can be classified as an acid. A second definition is that an acid is a proton donor
and a base is a proton acceptor (the Bronsted definition). It is often convenient to focus on
the molecule that is donating or accepting a proton, making this view of acid–base useful for
our study of amino acids.
Figure 5.9 shows a titration curve of glycine, in which we imagine starting with a fully pro-
tonated molecule and adding increasing amounts of the base NaOH. Notice that the shape
of this titration curve resembles that of a single dissociable group, but repeated so that two
rather than one equivalent of NaOH is required to remove both protons from the amino acid.
There are two buffering regions, corresponding to the carboxyl group and the amine. The
FIGURE 5.9 Titration curve for glycine. Glycine has two titratable groups; the titration curve
appears as if two separate dissociable groups were titrated sequentially. The flat regions centered
at B and D are also called buffer regions because modest increases in acid or base have little effect
on the pH in those regions. The center points B and D extrapolate to the two pK values on the
pH scale.
86 5.4 Acid–Base Properties and Charge
center point of a flat region, extrapolated to the pH axis, corresponds to the pK value for a
dissociable group. All of the pK values are listed in Table 5.2.
Five regions of the curve are marked A through E. Abbreviated structures emphasizing
just the dissociable groups are shown in Figure 5.10. Structure A exists at a pH value well
below pK1. Both the carboxyl and the amine group are protonated. Thus, the carboxyl group
is neutral, while the amine has a charge of +1, and the molecule has a +1 charge overall. At
the opposite end of the titration curve, point E is in the pH region well above pK2. In struc-
ture E, the carboxyl group is dissociated and has a charge of −1, and the amine group is dis-
sociated and has a charge of zero. This structure overall has a charge of −1.
Point C in the center of the titration is well above pK1 but well below pK2. Since pK1 refers
to the carboxylate, it is dissociated and charged −1. Notice that this group bears the same
charge as the carboxylate in structures D and E, since a charge of −1 remains at all values of
increasing base (and pH) well above pK1. Consider now the amine group of structure C. It
is still well below the value of pK 2, so it remains protonated, just as the other structures that
are in even more acidic conditions, that is, at points A and B. Thus, while both dissociable
groups are charged, the net charge on the molecule is zero. This structure is known as a
zwitterion, from the German zwitter, meaning hybrid. A prior example of a zwitterion is
phosphatidylcholine in Chapter 3; in that case, however, the positive charge resulting from
the quaternary amine.
Points B and D are at the center of the buffering regions of the titration curve. At these
points, the pH values match the pK values. The buffering region, introduced in Chapter 2,
is the portion of the curve in which there is minimal change in pH with a change in added
base (or added acid if we reverse the direction of the titration). The operational description
of buffering is that the weak acid resists changes in pH. The reason for it is mathematically
FIGURE 5.10 Charged forms of glycine over its titration range. Different points taken from
Figure 5.8 are drawn for glycine in this “conceptual titration” figure. The molecule is fully pro-
tonated at a very low pH, having a charge of +1.0 (A). At a very high pH, the molecule is fully
deprotonated, having a charge of −1.0 (E). Form (C) has exactly zero charge in the middle point
between pK1 and pK2. The other forms have half-charges because half of the groups at the exact
titration midpoint – that is, at the pK – have a charge of zero, and half have a charge of −1/2 (for
B) or +1/2 (for D).
Box 5.1 The Deceptively Simple Mathematics of Buffering
Every molecule that contains a dissociable group is a buffer, and the buffering range is
about 1 pH unit below and above the pK of that group. As stated in the text, the small
change in pH in the buffering region results from similar concentrations of both forms. To
make this clearer, let us return to the Henderson–Hasselbalch equation for the dissocia-
tion of HA:
[B4.1] pH = pK + log
[A -]
[HA ]
The closer the two forms of the group are in concentration, the closer their ratio is to 1.
Since log 1 = 0, then the pH = pK. Suppose a small amount of NaOH is added to the solu-
tion. The concentrations will change by a small amount x:

[B4.2] pH = pK + log
[A -] + x
[HA ] - x
Since the numerator is larger and the denominator smaller, the log term is larger, and so
the pH increases, as we expect when we add base. However, the ratio is still nearly 1, so
long as the [A–] and [HA] are significantly larger than the amount x. Both forms will be
much greater than x in this buffering region, maintaining a relatively constant pH. As the
amount of x increases, this ratio increases more drastically; its addition to the numerator
and subtraction from the denominator ensures that the change will not be linear, but
increasing. When we assign the buffering region to changes within 1 pH unit of the pK, it
is somewhat arbitrary; clearly, the closer the pH and pK values are, the better the buffer-
ing response. The same reasoning applies to a basic group if we start the titration in the
opposite direction with a fully deprotonated species and add acid. In summary, the phe-
nomenon of buffering is a simple consequence of the mathematics of ratios.
simple: in this region, there are similar concentrations of the two forms of the dissociable
group; small changes in either form have only small effects on the pH (Box 5.1). At the
center of the first buffering region – point B – there is an exactly equal amount of –COOH
and –COO−. Since half of the species have a charge of zero and half have a charge of −1,
the overall charge of the group at this point is −1/2. A similar situation applies to the amine
group at point D. In this case, the equal amounts of −NH 2 and −NH3+ means that the net
charge will be +1/2.
5.4.2 INTERACTIONS BETWEEN THE α-CARBOXYLATE AND α-AMINE GROUPS
Since the alpha carbon of amino acids is connected to both an acid (carboxylic) and a base
(amine), the groups interact. There are two ways in which the groups interact: by induction
(a through-bond effect) and by an electric field (a through-space effect). Taking both into
consideration enables us to explain the unusual pK values of the α-carboxyl and α-amine
groups of amino acids.
5.4.2.1 INDUCTIVE EFFECT
The inductive effect is based on the concept of electronegativity applied to groups within
a molecule. Electronegativity applies to two atoms across a covalent bond: the atom that
attracts the electrons the most has a greater electronegativity value. We have used vector
notation for the water molecule in Chapter 2. Similarly, vectors expressing the electronegativ-
ity differences across a bond can be summed within a group rather than the whole molecule.
88 5.4 Acid–Base Properties and Charge
For example, in the case of a methyl group, the C is more electronegative than the H. All
three vectors of a methyl group sum to make this substituent have a net outward vector:
C
H
We call this group electron releasing. On the other hand, consider substituting one of
the H atoms for the very electronegative F atom. The net vector now points in the opposite
direction:
C
H
Whereas the difference between C and H is not very large: C (2.55) − H (2.2) = 0.35, the
difference between F and C is considerable: F (4) − H (2.2) = 1.8. As this is greater than the
sum of the other two C–H bonds, the net vector is inward. The CH2F group is therefore
electron-withdrawing, as it will pull electron density from the rest of the molecule. We can
place these groups in the context of a carboxylic acid, as illustrated by the structures below:
H
F
C O O C O
H H
C HC C
H H
O O O
A B C
The electron-releasing methyl group in A puts more electron density into the carboxyl
group. We can think of this as increasing the negative charge of the carboxylate. Thus, A
will more strongly attract a proton than it would in the absence of the methyl group (i.e.,
it will have a larger pK than structure B). In addition, this electron release by the methyl
group diminishes resonance stabilization of the carboxylate anion. The opposite situation is
presented in C. The withdrawing group allows a greater spread of the carboxylate electrons,
enhancing the resonance of the carboxyl group. It also diminishes the negative charge. This
group favors hydrogen ion release; structure C is a better Bronsted acid (and has a lower pK
value) than structure B.
We can make a similar argument for the amine group. In this case, we are considering
the conjugate acid strength of the ammonium group. We would expect an electron-releasing
methyl group to stabilize the positive charge on the ammonium group, thereby increasing its
pK; this is borne out in Table 5.3. We can also predict the opposing direction for an electron-
withdrawing group. The difluoro-substituted amine is listed in the table; it has a much lower
pK. Thus, removing electrons from the amino group makes it a better Bronsted acid. An
equivalent statement is: as electrons are removed from the amino group, that group has a
reduced ability to accept a proton (i.e., it is a weaker Bronsted base).
5.4.2.2 ELECTRIC FIELD EFFECT
A foundational concept in physics is the influence of a test charge on other charged species.
Chemical species that have charge are ions, and they influence other portions of the same
molecule in all directions. This influence is the electric field effect, a “through-space” effect.
It is starkly different from induction, which has a specific direction indicated by the marked
TABLE 5.3 Group pK Values

Acid pK
carboxyl
HCOOH (formic) 3.95
CH3COOH (acetic) 4.75
FCH2COOH (fluoroacetic) 2.6
Average COOH of aa 2.2
amine
NH3 (ammonia) 9.0
CH3NH2 (methyl amine) 10.7
F2CNH2 7.52
H2 7.8
O C
NH2
OCH3 (glycine methyl ester)

Average NH2 of aa 9.5 G COO–
NH2
NH2
F COO–
pK3 = 10.28 NH2
NH2.5(+1/2)
E COO–
NH2
NH3+
FIGURE 5.11 Inductive and through-space effects on amino acids. A generalized amino acid
(A) is assumed to have average dissociation constants for the amine and carboxyl groups, and is D COO–
pK2 = 8.9 –NH2.5
not influenced by the R group. (B) The isolated methyl-carboxyl group should have a pK close to
pH
acetic acid. (C) Charged form of the amino acid with opposite charges attracting through space. NH3+
(D) The isolatedmethyl-amine group should have a pK close to methylamine.

C COO–
vectors. Like induction, the electric field effect is stronger when the two groups are closer NH3+
together. The significant electric field effects for amino acids are those between the neighbor- NH3+
ing α-amine and α-carboxylate groups in their charged forms.
To appreciate the interplay between electrical and inductive effects, imagine an amino B –COOH0.5
acid in the form of Figure 5.11a. Setting aside the influence of the R-groups, we can take the pK1 = 2.2 NH3+
averages of the pK values for the α-carboxylates and α-amines from Table 5.3. The pK of the NH3+
carboxylate group adjacent to methylene (Figure 5.11b) should be similar to acetic acid, 4.75.
A potent through-bond effect would be the fluoroacetate example of Table 5.3: this pK is 2.6. A COOH
Yet the pK of the carboxylate is even lower: it is 2.2. This low value is due to the stabilization NH3+
of the carboxylate forming the zwitterion of Figure 5.11c. The local positive charge of the NH3+
amine stabilizes the negative charge of the carboxylate form, making the latter a much better
acid. We conclude that the anomalously low pK for the carboxyl group of an amino acid is
the result of the electric field effect. FIGURE 5.12 Charge forms
The other expression of the electric field effect is that of the α-carboxylate on the α-amine of lysine over its titration
group. From the standpoint of the fragment outlined in Figure 5.11d, the pK of the amine range. Similar to the forms of
should be 10.7. We have a good estimate of the inductive effect on this pK from the value glycine in Figure 5.8, lysine has
for glycine methyl ester in Table 5.3: this is 7.8, which is close to that of the difluoromethyl an extra dissociable group,
amine as shown in the same table. Yet, the average pK of the amine group is instead 9.5, three pK values, and thus
much closer to the methylamine value. This raising of the pK is due to the electric field effect, seven regions with charges
acting in opposition to the inductive effect. Despite the change in direction, the explanation ranging from +2 (acidic) to −1
is the same as for the carboxyl group: the attraction of the positively charged amine to the (basic).
90 5.5 The Peptide Bond
negatively charged carboxylate. The higher pK means the amine is a relatively weaker conju-
gate acid, preserving its charged form. The electric field effect is dominant over the inductive
effect.
If we peruse Table 5.2, we find the pK values of the α-carboxylate and α-amine values for
all of the amino acids are remarkably uniform and far from what we would expect from an
isolated acetic acid and methylamine. The greatest divergences occur among those which
have a dissociable R-group. To appreciate this, we next consider the general topic of multiple
dissociable groups within a molecule.
5.4.3 MULTIPLE DISSOCIABLE GROUPS
Several amino acids have dissociable groups beyond the α–carbon attached carboxyl and
amine functions. For example, acidic amino acids have an extra carboxyl group, and basic
amino acids have an extra amine. Any other functional group that can dissociate can be
treated similarly, such as the -SH group of cysteine. These R-groups are distant enough from
the α-amine and α-carboxyl, so they have similar pK values to those in isolated molecules.
For the R-groups of the amino acids in general, the electric field effects are not significant.
Lysine is an example of an amino acid with multiple dissociate groups, having two amines
and one carboxyl group. Its titration curve is very similar to Figure 5.9, except there is a third
segment, with yet another flat region surrounding the third pK. The major species are shown
in Figure 5.12. There are now three pK values and seven structures represented. Again, the
net charge of the molecule is simply the sum of the charges of each functional group. Like the
simpler amino acids, species close to a pK value have fractional charges. For example, when
the pH is equal to pK1, lysine has a charge of +1.5.
There is a unique pH value at which lysine has a charge of exactly zero. This pH value is
the isoelectric point, drawn as E in Figure 5.12. At its isoelectric point, the molecule will
not move under the influence of an electric field, which is the operational definition of the
isoelectric point. To calculate the exact pH value for which this occurs, we take the average
of the two pK values that surround this species:

(5.3) pI = ( pK 2 + pK 3 ) /2
where pI is the symbol for the isoelectric point. For amino acids that have just two dissociable
groups:
(5.4) pI = ( pK1 + pK 2 ) /2
Thus, to find the pI of any molecule, we first identify the species with a zero net charge and
take the average of the pK values surrounding it. While seemingly a straight forward calcula-
tion, the average computed here involves pK values rather than the K (dissociation equilib-
rium constant) itself. This concept is further explored in Appendix A1.3, under Geometric
Mean.
5.5 THE PEPTIDE BOND
The most common biological linkage of amino acids is between the α-carboxyl group of one
amino acid to the α-amino of another. Overall, this reaction is a dehydration, as shown in
Figure 5.13. The process of protein synthesis is complex, involving many different proteins
and ribonucleotides (tRNA, mRNA, and rRNA; see Chapter 16).
The bond itself is an amide, like the one in asparagine and glutamine. The amide bond
between amino acids is known as a peptide bond. The chemistry of the bond is analogous to
an ester, except that the peptide bond is more stable. The stability can, in part, be explained
by the resonant structures of the amide bond:
FIGURE 5.13 Peptide bond formation. This is the formal view of the formation of the peptide
bond, which is a dehydration reaction, joining a carboxylic acid of one amino acid with the
amine of another.
(5.5)
The first charged form indicates a double bond between the C and N atoms, accounting for
the fact that there is little free rotation between these atoms. It is possible to draw the alter-
native resonance form:
C N
which shows bonds intermediate between single and double, a greater spread of electrons,
and an elimination of the charge forms.
The second charged form shown on the far right of Equation (5.13) accounts for extensive
intramolecular hydrogen bonding that exists in large strings of amino acids involving the
oxygen and hydrogen of the peptide bond (Figure 5.14).
The partial double bond character of the amide also prevents free rotation about this
bond. The lack of rotation about the C–N bond restricts the possible three-dimensional
arrangements of peptide-bonded structures.
Once peptide bonds are formed, the component amine and carboxyl groups no longer
have acid–base characteristics. Only the amine group at one end of the molecule and the
carboxyl group at the other end are dissociable, along with the side chains (the R groups
attached to the alpha carbons). Therefore, the ends of the chain are distinct: there is one
amino end (the N-terminus) and a carboxyl end (the C-terminus). The polymer itself has
distinctive properties, which vary with the length of the chain and the composition of the
R-groups.
92 5.6 Peptides and Proteins
FIGURE 5.14 Hydrogen bonding between peptide bonds.
FIGURE 5.15 Angiotensin II and vasopressin.
5.6 PEPTIDES AND PROTEINS
Short chains of amino acids, roughly up to 25 amino acids, are called peptides; longer chains
are called proteins. The cutoff between these is somewhat arbitrary, but there is a functional
difference. Peptides have flexibility in their structure, due to rotations about single bonds, a
feature they share with other relatively small molecules. However, proteins have a more fixed
behavior in solution as a result of intramolecular interactions. Three-dimensional structures
are characteristic of other polymers, such as polysaccharides. Large molecules that occur in
biological systems are called macromolecules.
Two examples of peptides are both hormones that regulate blood pressure; these are dia-
grammed in Figure 5.15. Angiotensin II is a linear peptide, whereas vasopressin is cyclic,
resulting from a bridge formed between the sulfhydryl groups of two cysteines. While the
latter is thereby more constrained in its structure, both have no fixed structure in solution
and change their conformation upon binding to their hormone receptors. Many peptides are
formed by proteolysis extracellularly, such as in the stomach and intestine (Box 5.2).
Box 5.2 Protein Digestion
Proteins are converted to amino acids in a two-step process in humans. First, they are
precipitated by the strongly acidic environment of the stomach and then exposed to
pepsin – a protease or enzyme that hydrolyzes peptide bonds. After a few hours in the
stomach, most of the proteins are converted to peptides, which enter the intestine. In
the intestine, they are subjected to further proteolysis by many distinct proteases that
produce amino acids, dipeptides, and tripeptides. The peptides are further hydrolyzed by
enzymes attached to intestinal epithelia (lining cells of the intestine, called enterocytes)
to free amino acids, which are then transported into the epithelia and, ultimately, into the
bloodstream. In short, proteins enter the stomach, and peptides leave it.
Larger protein molecules can be said to have polymer behavior, the result of intramolecu-
lar bonding. The description of protein structure is simplified by considering it in terms of
distinct levels.
5.7 LEVELS OF PROTEIN STRUCTURE
The levels of protein structure form a hierarchy: primary, secondary, tertiary, and quaternary.
Primary structure is the sequence of amino acids in a linear protein chain. Secondary
structure refers to a regular, repeating structural element that is formed when a segment
of the chain folds in three dimensions. Tertiary structure refers to the entire three-dimen-
sional arrangement of a protein chain in three dimensions. Finally, quaternary structure
is the three-dimensional arrangement of multi-subunit protein chains that associate non-
covalently. A separate level of protein structure emerged after this hierarchy was conceived,
falling between secondary and tertiary: the domain. A domain is a combination of second-
ary structural elements within a protein chain with a specific function, such as a binding
pocket of enzymes.
5.7.1 PRIMARY STRUCTURE
The primary structure of a protein is defined as its amino acid sequence. For peptides, the
primary structure is the complete description of the molecule. However, proteins have a
definite three-dimensional structure, requiring further levels of description. The experimen-
tal determination of the primary structure is usually obtained from the coding sequence of
the messenger RNA for the protein. In the past, the primary structure was established by
degrading proteins with proteases that have known preferences for cleavage sites. Analysis of
the protein fragments from different proteases enabled the solution of the primary structure.
This procedure is still used for small peptides that result from proteolysis.
The primary structure of a protein should dictate its complete spatial arrangement,
assuming that the chain folds into its lowest-energy conformation. The theoretical prediction
of that three-dimensional structure, however, is far from simple. Despite decades of study
and increasingly sophisticated computer power, it is still not possible in most cases to predict
that structure computationally.
5.7.2 SECONDARY STRUCTURE
Secondary structures are single elements of a regular, repeating three-dimensional struc-

ture in proteins (Figure 5.16). The two main secondary structures are the α-helix and the
β-sheet. Within a protein strand, they are joined together by stretches of amino acids with
no recognizable structure known as loops or random coils.
94 5.7 Levels of Protein Structure
FIGURE 5.16 Secondary structures.
5.7.2.1 α-HELIX
Within the spiraling chain of the α-helix (Figure 5.17), hydrogen bonds form between the N
and O atoms of aligned peptide bonds. The α-helix inner core is entirely hydrophobic for the
same reason discussed for cellulose: all of the hydrogen bonds are satisfied intramolecularly.
None of the hydrophilic groups are free to hydrogen bond with water. The nature of the
R-groups determines the water solubility of the entire segment. The latter extends to the out-
side of the spiral, as evident from Figure 5.16b. If the R groups are also hydrophobic, then the
entire helical segment is hydrophobic. It is common for a membrane-bound protein to have
the portion that passes through the membrane composed entirely of hydrophobic α-helically
arranged amino acids. Hydrophobic stretches of protein are critical for membrane insertion
as the membrane is itself hydrophobic. Two types of membrane proteins are indicated in
Figure 5.18. In one, the protein makes one pass through the membrane. The other protein
makes multiple passes, a typical arrangement for receptor proteins and ion channels.
Certain amino acids favor helix formation, whereas others hinder it. For example, hydro-
phobic amino acids or isolated charged side chains generally permit helix formation. On the
other hand, electrical repulsion due to neighboring charges (say, from multiple aspartate
residues) prevents the regular spiral from forming: these are helix breakers.
5.7.2.2 β-SHEET
The β-sheet is also known as the β-pleated sheet as it resembles a corrugated plate (Figures 5.16
and 5.19). The β-sheet is stabilized by hydrogen bonds between backbone peptide bonds, as
in the α-helix. However, the R groups cannot be excluded from an inner core of peptide
bonds in the β-sheet. Furthermore, not all of the potential hydrogen bonds are formed in the
β-sheet. The alignment of the chains can occur in two different ways. If the strands run in the
same direction, then the sheet is said to be parallel; if they run in opposite directions, then
it is said to be antiparallel (Figure 5.20).
5.7.3 DOMAINS
Specific combinations of secondary structures that exist in many different proteins are called
domains. Domains exist between the secondary and tertiary levels of structure. The reason
for the interest in domains is that they often correspond to a specific function. The terms
motif and supersecondary structure are used for the same class of intermediate structures
(see Box 5.3).
FIGURE 5.17 The alpha helix. Several amino acids are numbered 1 through 9. The repeated
backbone of CαC′N CαC′N CαC′N… where C’ represents the carbonyl carbon that makes up the
spiral of the helix. The intrachain hydrogen bonds between the carbonyl oxygen (red) and the
NH (blue) are indicated as dotted red lines. Note all possible hydrogen bonds are engaged, mak-
ing the entire internal segment hydrophobic.
FIGURE 5.18 Membrane spanning alpha helices. In A, the α-helix spans the membrane a sin-
gle time; certain hormone receptors have this structure. In B, the protein threads the membrane
several times so that multiple α-helices are present within the membrane.
FIGURE 5.19 The beta sheet. Hydrogen bonding in the beta-sheet stabilizes this structure
into a pleated arrangement.
FIGURE 5.20 Antiparallel and parallel beta sheets. The schematic arrangement of both types
of beta-sheets is indicated, with large arrows representing the beta-sheet portion and curved
lines representing random coil connections between them.
Box 5.3 Domain Names
We have used the term domain to indicate a structural level between secondary and ter-
tiary that combines secondary structure into a functional whole. Others have used differ-
ent terms such as motif and supersecondary structure, which also refer to a structural
level between secondary and tertiary. While it is possible to devise distinctions in the use
of these terms, others use them interchangeably. As there are no formally recognized
distinctions between the terms, we treat them as synonyms. It should be noted that the
identities of the various protein domains themselves have no systematic classification as
yet, even though domains have been known for 30 years.
The TIM domain (Figure 5.21) is donut-shaped, with an inner portion composed of a
β-sheet and an outer portion of α-helices. The inner portion also exists on its own in some
proteins, where it is known as a β-barrel. The TIM domain was named for the enzyme tri-
ose isomerase, in which this domain is a portion of the active site. This domain is the most
common one found in proteins.
The SH2 domain and the PTB domain, illustrated in Figure 5.22, both bind phosphor-
ylated tyrosine residues in proteins serving as part of signal transduction systems. The
FIGURE 5.21 The TIM domain. This domain has a central region of β-sheet and an outer ring
of α-helices. The most common of the domain structures is named for triose phosphate isomer-
ase, the enzyme in which it was first discovered.
FIGURE 5.22 Phosphotyrosine residue binding domains. Both the SH2 (left) and PTB (right)
domains recognize the same structure: a phosphorylated tyrosine residue of a protein.
SH2 domain was first found in the src or sarcoma virus protein, from which the name Src
Homology was derived. The PTB domain stands for Phosphotyrosine Binding Domain.
Figure 5.23 illustrates the NAD binding domain, which binds an important redox trans-
fer cofactor used by a large number of enzymes. The EF hand domain (Figure 5.24) selectively
binds Ca2+ ions in both enzymes as well as dedicated binding proteins, such as calmodulin,
and the endoplasmic reticular protein Stim1. The EF-hand domain (about 40 amino acid
residues) has a helix-loop-helix design that selectively and tightly binds Ca2+. Some proteins
bind calmodulin as a subunit. In this way, an elevation in cellular Ca2+ can be sensed by a
protein that does not itself bind Ca2+: the ion binds the EF hand domain, which alters the
structure of calmodulin and subsequently a target protein. For example, the protein Stim1
binds Ca2+ within the endoplasmic reticulum and serves as a sensor to control the refilling of
this subcellular space as a function of the existing Ca2+ concentration.
5.7.4 TERTIARY STRUCTURE
The three-dimensional structure of an entire protein chain is called the tertiary structure.
This structural level includes all aspects of the lower-level structures. We may also view
the tertiary structure as a collection of domains, joined by random coils (also known as
loops). For many proteins, this is the final structure level, as they consist of only a single
chain.
FIGURE 5.23 The NAD binding domain. This domain binds the cellular redox cofactor NAD+.
Two examples of a complete protein chain are myoglobin and adenylate cyclase:
Myoglobin is an oxygen-binding protein discussed later in this chapter. It is formed entirely

by α-helices connected by random coils. Adenylate kinase (Chapter 8) is an enzyme involved
in the interconversion of adenine nucleotides. This protein consists of both α-helices and
β-sheets.
5.7.5 QUATERNARY STRUCTURE
Proteins that consist of more than one chain have quaternary structure. The individual
subunits are typically bound together non-covalently, often through charge interactions or
FIGURE 5.24 The EF hand domain. This is a Ca2+ binding domain that occurs in proteins that
selectively bind this ion. The flexible joint between the helices allows movement when Ca2+ is
bound, triggering a response to a change in [Ca2+].
hydrophobic interactions at the interface of the subunits. The individual protein chains are
called monomers; the entire protein may contain two, three, four, and, occasionally, more
monomers. Some proteins contain identical monomers, which occurs in many enzymes.
Other proteins are composed of different subunits, which may vary only slightly, as in hemo-
globin, or dramatically, as with most ion channels.
In proteins with quaternary structure, an alteration in the conformation of one subunit
may affect a neighboring subunit, an important regulatory feature. However, many proteins
possess quaternary structure and yet have no known regulatory function.
With this final level of protein structure, we have a complete description of a protein’s
spatial arrangement. However, we have not yet considered the basis for the assumption that
the primary sequence determines all higher levels of structure. We address this topic next.
5.8 PROTEIN FOLDING
The enzyme ribonuclease (RNase) is a digestive enzyme secreted from the pancreas. The
enzyme is commonly used to study protein structure as it is relatively easy to purify and is
exceptionally stable. An experiment conducted in 1959 by Christian Anfinsen addressed the
question of whether the primary structure of a protein determines the three-dimensional
structure. The experiment is illustrated in Figure 5.25. First, the enzyme was heat-inactivated
and then allowed to cool. After cooling, the preparation regained enzyme activity, demon-
strated by its ability to hydrolyze the phosphodiester bonds in RNA. As this was a purified
system, no other components were needed for the renaturation. Evidently, only the amino
acid sequence itself was necessary to achieve the proper three-dimensional structure.
The discovery of the bacterial protein GroEL (Figure 5.26) in the 1970s enhanced our
understanding of protein folding. As proteins are being formed, GroEL helps them assemble
100 5.9 Oxygen Binding in Myoglobin and Hemoglobin
FIGURE 5.25 Refolding of ribonuclease. The active conformation of ribonuclease (RNase) is

disrupted by heat and restored by subsequent cooling. Activity is determined by measuring its
ability to cause rupture of the phosphate bonds in an RNA chain.
FIGURE 5.26 GroEL segment. Side and top views of a segment of the GroEL protein, a portion
of a chaperone that assists proteins in folding as they are synthesized. Seven of these are stacked
on top of each other to make half of the chamber; two of these are a complete GroEL. Once a
newly synthesized protein is ensconced in this assembly, a smaller protein (GroES) forms a cap;
this protein is also essential for accelerating protein folding.
correctly. Thus, while proteins can refold in vitro, the rate of this process is generally inade-
quate within cells. GroEL and similar proteins, called chaperone proteins, serve to catalyze
the folding process within the cell. The figure shows a portion of the active assembly; several
of these rings stack and form a cage within which correct protein assembly is accelerated.
5.9 OXYGEN BINDING IN MYOGLOBIN AND HEMOGLOBIN
Myoglobin and hemoglobin are two proteins that bind oxygen in mammals. They have very
similar protein structures, except that myoglobin is a single chain, and hemoglobin has four
monomer units. Myoglobin has a molecular weight of about 17,000 and contains a tightly
bound molecule of heme. The function of heme is to bind oxygen reversibly. Hence, the
protein itself only indirectly binds to oxygen, relying on this smaller molecule. In general, a
small molecule that is bound to a protein to assist its function is called a prosthetic group.
The structure of heme (Figure 5.27) shows four linked aromatic rings; each has a nitrogen
atom chelated to a central Fe2+. The word chelate is derived from claw; the multiple attach-
ment points of the electron-donating organic molecule hold Fe2+ tightly in place. The bond
between the nitrogen and the iron ion is known as a coordinate covalent bond or dative
bond. Like any covalent bond, the electrons are shared between two nuclei, here between
FIGURE 5.27 Heme with a central ferrous ion. Four of its six ligand positions are shown, che-
lated to N atoms of linked aromatic rings. Two other empty orbital positions of Fe2+ are shown
above and below the plane of the page. One binds a histidine residue of hemoglobin; the other
reversibly binds oxygen.
nitrogen and iron. However, all of the electrons are donated by the nitrogen, just like an
ionic bond. Nonetheless, the dative bond has a direction, unlike the electrostatic interaction
of an ionic bond. Dative bond formation can be considered as an acid–base reaction, under
the Lewis definition of acid–base. Fe2+ is the Lewis acid (electron pair acceptor), and the
nitrogen is the Lewis base (electron-pair donor). The symbol for the dative bond, also shown
in Figure 15.27, is an arrow between the nuclei, with the arrowhead pointing to the electron-
deficient nucleus (iron in this case). Fe2+ has a coordination number of six; this means that
it forms six directed bonds to electron-rich atoms. The four orbitals illustrated are in one
plane; the other two (not shown in the figure) are above and below the plane. One of these
is bound to a histidine residue of the myoglobin protein; the sixth is occupied by oxygen (in
oxygen-bound myoglobin) or is unoccupied (in free myoglobin). The oxidation state of iron is
fixed in myoglobin at +2; that is, it is not redox-active.
Abbreviating myoglobin as Mb, we can express oxygen binding to the protein as an
equilibrium:

(5.6) Mb × O2 Mb + O2
(5.7) K diss =
[Mb][O2]
[Mb.O2]
This equilibrium is not typical, as it involves molecules both in the gas phase and the liquid
phase. Two modifications make this experimentally useful. First, since oxygen is a gas, in
place of concentration, we use the partial pressure, PO2. Thus, the gas is measured by the
pressure it exerts in the gas phase in equilibrium with the solution. Secondly, rather than
using the equilibrium constant, we use a related measure: the fractional saturation of Mb
with oxygen. Fractional saturation is symbolized as the variable Y and defined as:
Y=
[MbO2]
(5.8)
[Mb] + [Mb.O2]
As indicated by this equation, Y is the ratio of oxygen-bound Mb to total Mb species (oxygen-
bound and free), and ranges from 0 to 1. The graph of Y versus PO2 is typical of any ligand
binding to a receptor, showing a curve that rises monotonically and saturates at high oxygen
content (Figure 5.28). Half-maximal binding occurs at about 5 mm Hg. Myoglobin stores
oxygen in muscle cells and releases it as prevailing levels become depleted. Oxygen release
102 5.9 Oxygen Binding in Myoglobin and Hemoglobin
FIGURE 5.28 Oxygen saturation curve for myoglobin. Y represents the fraction of myoglobin
bound to oxygen to the total myoglobin; PO2 is the partial pressure of oxygen. The shape of the
curve is typical for any simple binding that obeys a single equilibrium.
FIGURE 5.29 Model of cooperativity in binding of oxygen to hemoglobin. The subunits of

hemoglobin are represented as circles (low affinity) or squares (high affinity). Upon binding oxy-
gen, the subunits switch to a new conformation that is high affinity. Subsequently, neighboring
subunits are affected and switch to high-affinity sites.
is accomplished simply through the equilibrium presented above. Myoglobin is present in

humans but particularly abundant in diving mammals such as whales and seals.
The protein hemoglobin has quaternary structure, having four monomer units of two
types. Nonetheless, all these monomers are very similar in size and structure to myoglobin.
Each has a bound heme group, and each can bind a molecule of oxygen. In hemoglobin, the
binding of oxygen to each subunit changes the subsequent binding of oxygen to another
subunit. This behavior is called cooperativity, as opposed to independent monomer bind-
ing, which is called non-cooperative. A mechanism is shown in Figure 5.29 in which the
conformation of the low affinity monomers in hemoglobin are represented as spheres; high-
affinity monomers are represented as blocks. As oxygen binds, it alters the conformation of
the monomers. Subsequently, neighboring subunits change their conformation even in the
unbound state, and the entire protein now binds, ensuing oxygen molecules with greater
affinity. This phenomenon is called positive cooperativity.
While it is possible to write equilibria for hemoglobin binding to multiple oxygen mol-
ecules, the equations are complex. We will examine the experimentally observed behavior
of oxygen binding to hemoglobin expressed as the fractional saturation (Y) as presented in
Figure 5.30. The “S-shape” of this figure is characteristic of positive cooperativity. At low PO2,
there is minimal oxygen binding to Hb. As oxygen level is increased (equivalently, increased
PO2), not only does Y increase, but the rate of rise increases. This tendency is merely the graph-
ical expression of positive cooperativity. As PO2 is increased even further, the rate falls off as
saturation is reached (i.e., no more binding sites on the protein remain), and Y approaches 1.0.
FIGURE 5.30 Oxygen saturation curve for hemoglobin. As a consequence of cooperativity,

the binding represented as Y increases as more oxygen molecules occupy hemoglobin subunits.
Binding falls off once saturation is reached.
FIGURE 5.31 Myoglobin versus hemoglobin. Both saturation curves are plotted together. Mb
(myoglobin) has a much lower midpoint for binding than Hb. Only Hb binding is cooperative.
Comparing curves for both oxygen-binding proteins (Figure 5.31), it is apparent that Mb
binds O2 tighter than Hb; that is, the midpoint occurs at a far lower PO2 for Mb. The physi-
ological role of Mb is to provide O2 when its concentration is very low for intracellular use, so
its tight binding is appropriate. Hb, the major protein of the red blood cells, is saturated with
oxygen in the lung capillaries (PO2 of about 100 mm) and will release this oxygen to equili-
brate with the much lower oxygen content of the tissue capillaries (PO2 of about 40 mm). This
equilibrium is the fundamental regulation of oxygen uptake and delivery.
There are modifications to the binding of oxygen with Hb. One of them, called the Bohr
effect, is the effect of protons on Hb binding to O2 (Figure 5.32). The entire curve is shifted
to the right under acidic conditions. For example, an increased anaerobic metabolism leads
to lactic acid production, a weaker binding of O2 to Hb, and an increased release of oxygen.
There is also an adaptation which occurs at high altitude. In this condition, the red blood
cell produces the molecule 2,3-bisphosphoglycerate, which lowers the binding of Hb to O2
in a manner similar to protons (Figure 5.32). Oxygen release from Hb in the presence of
2,3-bisphosphoglycerate provides more to the tissues, alleviating the problem of lower oxy-
gen content availability to tissues at high altitude.
5.10 OTHER BINDING REACTIONS INVOLVING PROTEINS
Aside from Mb and Hb, equilibrium-binding to many other proteins serves other essential
physiological functions. They vary in terms of their location (e.g., intracellular versus extra-
cellular) and strength of binding, as indicated by the equilibrium constant. Hemoglobin and
104 5.10 Other Binding Reactions Involving Proteins
FIGURE 5.32 Altering the hemoglobin saturation curve. Substances that decrease the bind-
ing of oxygen to hemoglobin, such as protons (H+) or BPG (2,3-bisphoshoglycerate) shift the
curve to the right. The line drawn at 40 mm of Hg shows that for the same value of PO2 the shifted
curve will be in equilibrium with a less saturated hemoglobin. In other words, hemoglobin will
hold on to much less oxygen at the same oxygen partial pressure in the presence of increased
acidity or BPG; this delivers more oxygen to tissues.
myoglobin, the oxygen binding proteins just described, are intracellular binding proteins.
Here we consider further examples of binding proteins.
5.10.1 EXTRACELLULAR BINDING PROTEINS
Serum albumin is a protein (67,000 D) composed of a single strand that binds hydrophobic
molecules in a somewhat nonspecific way. It is a major binding protein for fatty acids that are
transported from their site of release from the adipocyte to their site of oxidation by several
cell types, including those of liver, skeletal muscle, and heart.
An example of a more selective binding protein is ferritin, which binds only iron and
enables this ion to move through the circulation. In addition, the complex of iron with fer-
ritin can be considered a storage form of iron.
5.10.2 CELL SURFACE BINDING PROTEINS
Many proteins are embedded in the plasma membrane and have binding sites for molecules
in the extracellular space. For example, hormone receptors bind blood-borne hormones to
their plasma membrane-bound receptors, which respond by initiating signaling systems. In
some cases, the membrane proteins are part of the immune system to bind receptors of other
cells, such as T lymphocytes. Transporters are yet another class of cell surface protein that
provide extracellular molecules a means of entry into cells. A further type of membrane-
bound protein serves as a tether to connect cells within a tissue to each other and to extra-
cellular fibers.
5.10.3 INTRACELLULAR BINDING PROTEINS
There are numerous intracellular binding proteins. Some play a similar role to cell surface
proteins. For example, lipid soluble hormones cross the plasma membrane and subsequently
bind to intracellular sites, such as thyroid hormone, vitamin D, retinoids (vitamin A deriva-
tives), and steroids. Calreticulin is a distinct binding protein located exclusively in the lumen
of the endoplasmic reticulum, where it serves to bind calcium ions at equilibrium and main-
tain a very high free concentration of nearly 1 mM within that space. Finally, there is a large
class of signal molecules that are binding proteins. For example, G-proteins (which bind
guanine nucleotides) transduce membrane-bound receptors; actins bind motor proteins; and
nucleic acid binding proteins regulate the ultimate formation of new proteins.
5.11 PROTEIN PURIFICATION AND ANALYSIS
The rise of biochemistry as a distinct area of chemistry coincided with methods to purify
and analyze proteins. Accordingly, we conclude this chapter with a discussion of protein
methods.
5.11.1 PURIFICATION
The first requirement of protein purification is an extraction from its natural environment
with its properties intact. Subsequently, the extract is subjected to a fractionation process
that divides the material into unequal portions, ultimately producing just the protein of
interest.
Throughout the process, a specific measurement of the protein is required. For example,
enzymes are usually measured by analyzing the disappearance of substrate or appearance of
the product. All such methods – called assays – must also be simple to perform since they
will have to be repeated hundreds of times in the course of the purification.
Several precautions are commonly taken to minimize protein denaturation during extrac-
tion and purification. These include performing experiments in a cold room, inclusion of buf-
fer solutions to prevent extremes of pH, addition of reagents to avoid oxidation of cysteine
side groups, and the inclusion of protease inhibitors to prevent rupture of the peptide bonds.
The extraction procedures may use physical methods, such as a blender to tear the tissue
and release intracellular content or detergents, to chemically solubilize the cell membranes.
Once a solution is produced that contains the target protein, fractionation can proceed. The
most commonly used separation methods are based on differences in solubility differences,
size, and charge. As a simple example, salt precipitation is a method for separating proteins
by differences in solubility that stems from charge differences. Different concentrations of
salts (usually ammonium sulfate is used as it preserves protein conformation) precipitate dif-
ferent proteins, as they vary in total charge.
Most purification schemes include a form of chromatography (Box 5.4). This pro-
cedure is defined as a separation of components due to their different affinities towards
two distinct phases: a mobile and stationary phase. For example, in ion-exchange column
Box 5.4 Word Origins: Chromatography
In 1901, the Russian botanist Mikhail Tsvet improved upon prior separation methods by
filling a cylinder with a support material, loading plant leaf extracts, and subsequently
eluting fractions with organic solvents. Several distinctly colored compounds emerged,
leading Tsvet to name the process chromatography, which means color writing. Modern
chromatography refers to any of a wide array of methods that effect a separation by par-
titioning molecules between mobile and stationary phases.
While the word chromatic may appear to apply strictly to colors, it does have another
origin. In early Greek society, it was a description of elaborate rhetoric. In early Greek
usage, the word was applied to music as one of three types of tetrachords (the others
being diatonic and enharmonic). Today, the word remains in music, where the chromatic
scale includes semitones (sharps and flats); the diatonic scale does not. Thus, the word’s
richness is commensurate with the richness of the chemical technique and its ongoing
modifications.
106 5.11 Protein Purification and Analysis
FIGURE 5.33 Ion exchange chromatography. The column is shown as a matrix of solid mate-
rial (called a gel) suspended in a buffer. In (a) a cell extract representing a mixture of proteins is
added to the top. Subsequently, a continuous flow of buffer fluid is passed through the column.
Fluid emerging from the bottom is collected in tubes. The expanded view (b) shows differently
charged proteins adhering with different strengths to the gel. This interaction alters the time
with which they elute, thereby achieving a separation. As in any chromatography, there is a sta-
tionary phase (the gel) and a mobile phase (the eluting buffer).
chromatography (Figure 5.33), negatively charged protein molecules added to the mobile
phase are retarded by the stationary phase to different degrees and emerge from the column
in different fractions. In size exclusion chromatography, proteins are retarded by various
sized cavities in the stationary phase beads, so large proteins leave the column first. In all
cases of chromatography, several modifications are possible. For example, it is possible to
vary the stationary phase by making much smaller beads and forcing fluid flow under pres-
sure, called high-pressure liquid chromatography (HPLC). The sample and carrier fluid
can be converted into a gas phase in gas–liquid chromatography. A selective binding
may be achieved by having a molecule known to selectively attach to the protein in affinity
chromatography.
5.11.2 ANALYSIS
Proteins are typically measured and characterized by their interaction with light, or more
generally, radiation. For example, due to aromatic side chains such as tyrosine and trypto-
phan, proteins absorb ultraviolet radiation. Light absorbance provides an estimate of the
total amount of protein present. A more elaborate analysis is possible with polarized light to
determine protein conformation in solution.
The response of unpaired atomic nuclei to a magnetic field can be used in nuclear mag-
netic resonance (NMR), a technique of growing importance in assessing protein struc-
ture. While NMR has long been used for small molecule identification, applications of more
sophisticated instrumentation and computer analysis have led to an increased resolution in
structural studies of ever-larger proteins.
Bombarding crystallized proteins with X-rays produces a pattern that, after mathematical
manipulation, provides a high-resolution picture of the molecule, albeit in solid form. Protein
structures in the solid phase are likely somewhat different from those in solution phase, as
this method requires a solid crystal for analysis. The method of x-ray crystallography has
now been extended to increasingly large proteins and even membrane-bound proteins such
as ion channels.
Mass spectroscopy is another method borrowed from chemical analysis of small mol-
ecules and applied to protein analysis. Here, protein fragments are vaporized to a gas phase,
and the fragments are separated in an electromagnetic field according to mass.
A commonly used method to determine the molecular weights and subunit composition
of proteins is electrophoresis. This is a form of chromatography in which the mobile phase
is driven by the presence of an electric field imposed by electrodes and the stationary phase a
gel composed of a polymer, commonly acrylamide. This method can also be used to identify
various proteins present in a sample and as a test of purity. In some instances, it is used as
a method of purification itself, as it performs a separation, and the purified protein can be
extracted from the gel.
Much of what we have been considering in the present chapter will lead us to the next. For
example, the binding equilibrium behaviors of myoglobin and hemoglobin have analogies in
the behavior of the two major classes of enzymes: the corresponding diagrams even look very
much the same. It is critical to appreciate the distinction: in binding, we are witnessing equi-
librium behavior, but in enzyme action, the system is removed from equilibrium. We next con-
sider the action of the group that represents over 90% of all the known proteins: the enzymes.
SUMMARY
Amino acids are the protein building block; 20 of them are naturally occurring in proteins.
These may be divided between water-soluble and hydrophobic, further divided into acid–
base properties, or functional group chemistry. The latter include hydroxyl groups of differ-
ent types and the sulfhydryl groups that may react together, oxidizing to a disulfide linkage.
All amino acids have multiply charged groups at physiological pH; at least one amino and one
carboxyl group. Thus, varying pH produces multiple charge possibilities. At one specific pH,
the charge is zero; this is the isoelectric point. Joined together, the amine and carboxyl group
of two separate amino acids form a peptide bond. When small numbers (less than about 25)
of amino acids are so linked, the resulting molecule is called a peptide. These molecules still
display “small molecule” behavior. However, once they are larger, they assume the behavior
of polymers and are called proteins. The structure of proteins is formally divided into levels:
primary (the sequence of the amino acids); secondary (a recognizable unit of structure such
as an α-helix or β-sheet); tertiary (the three-dimensional structure of a protein strand); and
quaternary (the overall structure of multi-subunit proteins). An intermediate level of struc-
ture, lying between secondary and tertiary, associated with a known biological function such
as binding to a specific molecule, is called a domain. The overall formation of proteins into
an active three-dimensional form is called folding. While the information required to fold
correctly is inherent in the primary structure, cells have proteins that assist and accelerate
108 Review Questions
the process called chaperones. An example of protein function is the binding of oxygen to
myoglobin, which adheres to a simple equilibrium model. Hemoglobin is an analogous oxy-
gen-binding protein but has four separate subunits, each very similar to the structure of
the myoglobin monomer. The binding of oxygen molecules to hemoglobin is cooperative.
This term means that as more oxygen molecules bind Hb, the affinity shifts. Subsequent
oxygen molecules bind tighter, producing an S-shaped binding curve. This type of oxygen-
binding behavior provides a greater response of oxygen release than single-site binding,
which is appropriate for the oxygen delivery role of Hb. Several other proteins function by
equilibrium-binding, including hormone receptors, intracellular regulators, and structural
proteins. Protein purification and analysis methods for proteins were modifications of those
first developed for small molecules. A widely used purification method is chromatography,
the separation of proteins by their relative affinities towards a mobile phase and a stationary
phase. Several analysis methods are applied to proteins, including NMR (nuclear magnetic
resonance) which senses the spin of atomic nuclei; mass spectroscopy, in which proteins are
fragmented into charged ions and separated into different classes based on their masses;
and X-Ray crystallography, in which proteins are bombarded with high energy radiation and
their structure revealed by the scattering pattern.
REVIEW QUESTIONS
1. There can be other groupings for the amino acids; some are in multiple categories.
Suggest different groupings and identify those amino acids that can appear in more than
one category of these and the groups presented in the text.
2. Draw a titration curve for glutamate and show the major charge forms in a manner
analogous to the one for lysine in the text. What is its isoelectric point?
3. Suppose intracellular pH is 7.1. Most proteins are negatively charged at this pH.
a) What is the overall balance of acidic and basic side chains?
b) What type of ion-exchange chromatography would be most useful for separations?
4. An unknown amino acid is found to contain a hydroxyl group, and studies show it is
immobile in an electric field if the pH is exactly 5.655. What is it?
5. Polyamines are multiple amine-containing molecules derived from a non-protein amino
acid (ornithine) by loss of the CO2 group. One of these is putrescine, which has the
structure
NH 2 - (CH 2 )4 - NH 2
a) Give its titration curve, given the pK values of 9.35 and 10.8.
b) What is its pI?
c) Polyamines have at least two known functions: binding to DNA, and binding to K+
channels. Based on purely electrostatic effects, explain these biological activities.
6. The molecular weight of an amino acid is roughly equal to 100. This estimate allows a
quick estimate of the number of amino acids in a protein given its molecular weight or
the reverse operation. What is the approximate number of amino acids in hemoglobin?
Assuming a more accurate estimate of 115 as the molecular weight of an amino acid
residue, how would this change your answer?
7. Suppose the average concentration of a hormone in the blood is 1 nM. We can predict
the Kdiss for its receptor would also be about 1 nM. Explain why this is the case.
8. Give an example of a protein in which the interior must be (a) hydrophilic; (b) hydrophobic.
CHAPTER 5 ADDENDUM: ATOMIC CHARGED FORMS
Drawing charged forms of atoms within a molecule is a formal assignment that can con-
flict with chemical reality. As a prime example, consider a quaternary amine in which N is
assigned to have the charge +1:
( CH3 )4 N +
The problem is, N is more electronegative than the C it is attached to, so it is unreasonable for
it to actually be positively charged and C be zero. An improvement would be to assign each
carbon a quarter of a positive charge. However, then it would be less electronegative than
hydrogen! A resolution is to show the charge on the overall molecule, represented by enclos-
ing the molecule in brackets with the charge outside it.
In some cases, this type of charged form can be rewritten. For example, the middle reso-
nance form of the amide bond
can be rewritten as:
C N
so that the resonance is shown as spread out between the O, C, and N atoms. Here, both the
C–O and the C–N bonds are a bond and a half. This format has the advantage of eliminating
charge and emphasizing the delocalization aspect of resonance.
KEY TERMS
α-carbon
α-hydrogen
affinity chromatography
amide
amino acids
amino end
antiparallel
assay
β-barrel
Bohr effect
buffering regions
calmodulin
carboxyl end
chaperone protein
chelate
chromatography
cooperativity
coordinate covalent bond
coordination number
dative bond
domain
EF hand
electric field effect
electron releasing
electron-withdrawing
electrophoresis
Gas–liquid chromatography
helix breakers
110 Bibliography
heme
high-pressure liquid chromatography (HPLC)
isoelectric point
Lewis acid or base
loop
macromolecules
mass spectroscopy
monomers
motif
NAD binding domain
non-cooperative
nuclear magnetic resonance (NMR)
peptide bond
positive cooperativity
primary structure
prosthetic group
protease
protein
PTB domain
quaternary structure
random coil
secondary structure
SH2 domain
soft nucleophile
supersecondary structure
supersecondary structure
tertiary structure
TIM domain
umami receptor
x-ray crystallography
zwitterion
BIBLIOGRAPHY
Z. Aung, Li, J. Mining Super-Secondary Structure Motifs from 3d Protein Structures: A Sequence
Order Independent Approach. In: S.-K. Ng, H. Mamitsuka, L. Wong (Eds.) Genome Informatics:
Proceedings of the 18th International conference. Imperial College Press, London, 2007, pp.
15–26.
M. Aebi. N-Linked Protein Glycosylation in the Er. Biochim. Biophys. Acta, Mol. Cell. Res. 1833 (2013)
2430–2437.
E.V. Anslyn, D.A. Dougherty. Modern Physical Organic Chemistry. University Science, Sausalito, CA.
2006.
R.M.C. Dawson, D.C. Elliott. Data for Biochemical Research. Clarendon Press, Oxford. 2002.
D. Eisenberg. The Discovery of the Alpha-Helix and Beta-Sheet, the Principal Structural Features of
Proteins. Proc. Natl. Acad. Sci. USA 100 (2003) 11207–11210.
C.A. Fitch, G. Platzer, M. Okon, B. Garcia-Moreno E, L.P. McIntosh. Arginine: Its PK Value Revisited.
Protein Sci. 24 (2015) 752–761.
H. Hartley. Origin of the Word 'Protein'. Nature 168 (1951) 244. Hartley attributes the first use of the
word protein to Berzelius, along with isomerism, polymerism, and catalysis.
R. Irvine. Concepts of Protein Structure and Function. In: M.G. Ord, L.A. Stocken (Eds.) Early
Adventures in Biochemistry. Jai Press, Greenwich, CT. 1995, p. 165.
Databases: Protein Data Bank. http://www.rcsb.org; PubMed Conserved Domains, https://www.ncbi
.nlm.nih.gov/pubmed.
Enzymes 6
The word enzyme is German for in yeast. Near the end of the 19th century, the demonstra-
tion that a cell-free extract could cause sugar to ferment – as dramatically demonstrated
by the formation of bubbles of CO2 – was a blow to the vitalist theory that only intact cells
could carry out biological reactions. The extract was named zymase; today, we use the word
enzyme to describe a catalyst involved in one reaction.
A catalyst causes an increase in the rate of a reaction without affecting its equilibrium
position. To visualize the action of an enzyme, consider placing weights on a dual-pan bal-
ance (Figure 6.1). If weights are dropped onto both pans, they will oscillate until they set-
tle into their final equilibrium rest positions. If you intercede in this process just after the
weights are dropped using your hands as dampeners, the time needed to reach the final
state is much shorter. Yet the heights of the pans in their final equilibrium (rest) positions
are unaffected by this intervention. The steadying hands represent the action of an enzyme,
which increases the rate of approach to equilibrium (the rate of a reaction in both forward
and reverse directions) but does not affect the final state of equilibrium. An intriguing corol-
lary is that an enzyme has no effect on a reaction at equilibrium. Thus, the study of enzyme
kinetics is that of the time course approaching, but not having reached, equilibrium.
6.1 ENERGETICS OF ENZYME-CATALYZED REACTIONS
In any reaction, converting substrate to product requires intermediate states. Those inter-
mediates are always less stable – and thus of higher energy – than either the substrate or the
product. In the simplest theoretical case of one intermediate, we can speak of the transition
state, although invariably more than one exists. The difference in energy level between the
substrate and the transition state is called the activation energy. Energy input is needed to
move reactants up to the transition state’s energy level (Box 6.1)
A fundamental property of enzyme action is the lowering of the activation energy. We
can appreciate how this comes about by considering a progress curve for an uncatalyzed and
enzyme-catalyzed reaction side by side in Figure 6.2. Both reactions convert substrate S to
product P. In the uncatalyzed reaction, activation energy (Eact) is represented by a peak. We
need to input energy – usually by increasing the temperature – in order to bring the substrate
into an active intermediate state that can be converted to products. Enzymatic reactions pro-
ceed by a different pathway: the formation of an enzyme–substrate complex (ES). Because
the enzyme E can bind substrate S and form the ES intermediate, the reaction requires less
energy. Reactive groups contributed by the enzyme itself and fixed orientation of the sub-
strate enable the reaction to proceed to product with far less energy input requirement. For
this reason, we observe a greater number of peaks and a much lower Eact. Additionally, the
selective binding of S enforces specificity for the catalyzed reaction.
The chemical transformations of the substrate on the enzyme surface proceed as they
would in nonenzymatic processes (e.g., oxidation, proton transfer, or electron rearrange-
ment). Enzymatic reactions are orchestrated with precision. The orientation of the substrate
with ancillary groups – or even other substrates – benefits from selective binding to a spe-
cific region of the enzyme called the active site. The intermediates symbolized here (i.e., ES)
111
112 6.1 Energetics of Enzyme-Catalyzed Reactions
FIGURE 6.1 Equilibrium balance. A dual-pan balance models equilibrium. (a) Weights are
dropped on the balance, and (b) it oscillates about its true end state. (c) A pair of hands steadies
the balance, and (d) it achieves an equilibrium point, which is the same point that it would reach
without the hands in (d). The hands symbolize enzyme action.
Box 6.1 Activation Energy and Murphy’s Law
The concept of activation energy can be made intuitive by dispelling a common myth
known as “Murphy’s Law” which states that “if anything can go wrong, it will”. While
declaring this law generates a cynical acquiescence in listeners, it is in fact wrong – one
of the philosophical insights of chemistry. A more accurate law is: “most things can go
wrong, but don’t”.
The more stable state of a building is a pile of bricks rather than the very orderly
arrangement of the structure, yet we hardly fear an imminent collapse while we are
inside. This is because it would take a wrecking ball – a large investment of extra energy –
to reduce the building to a more stable arrangement. Similarly, virtually all reactions that
can proceed to a more stable form nonetheless require an input of great energy to get
them going. In the molecular world, we need to think of this as providing increased
movement of molecules (rotations, vibrations, translations) before the reaction can occur.
Thus, even though the final state may be more stable than the initial state, activation
energy is always required. Murphy was apparently an incautious investigator who rushed
to publication.
FIGURE 6.2 Energy of activation in uncatalyzed and catalyzed reactions. On the left is the high
energy intermediate of an uncatalyzed reaction between substrate and product. On the right, an
enzyme-catalyzed reaction shows an additional high-energy but stable region, the intermediate
enzyme–substrate complex (ES). The enzymatic reaction course has higher reaction barriers on
either side; the highest barrier that defines the Eact is lower than that of the uncatalyzed reaction.
Chapter 6 – Enzymes 113
will later be fleshed out in chemical terms. For now, our focus is on how the reaction rate
changes as the concentration of the substrate is varied.
6.2 THE ENZYME ASSAY AND INITIAL VELOCITY
Enzyme activity is measured as the number of moles of substrate converted to product per
unit time (e.g., moles per second). The laboratory determination of enzyme activity is called
an assay. As an example, consider the case of the enzyme alkaline phosphatase. This enzyme
is attached to the external surface of cells and catalyzes the dephosphorylation of various
substrates. The assay of enzyme activity uses the artificial substrate p-nitrophenylphosphate
(Figure 6.3). This compound is colorless, but the dephosphorylated product, p-nitrophenol,
is bright yellow in its anionic form. The increase in color can be followed continuously and
related to product concentration using a spectrophotometer (Box 6.2). The resulting time
course of product formation is shown in Figure 6.4. Note that the rate of product formation
steadily decreases with time. This decreasing slope results from a decrease in substrate con-
centration and a corresponding increase in product concentration with time.
Reaction velocity is computed as the ratio of a change in concentration to a change in
time. By convention, enzyme kinetics uses the portion of the curve indicated in Figure 6.4
as a broken straight line, representing the initial velocity v. This extrapolation avoids the
FIGURE 6.3 Phosphatase action on p-nitrophenolphosphate. The phosphatase enzyme cata-

lyzes the removal of the phosphate. The product p-nitrophenol equilibrates with its anion form,
which has a bright yellow color that can be quantified by spectrophotometry.
Box 6.2 The Spectrophotometer and Beer’s Law
One of the simplest analytical methods is the absorption of light energy; the instrument
used is called a spectrophotometer. Light energies from the visible and near-ultraviolet
range are isolated by a prism or a grating, passed through a sample, and then detected
by a sensitive phototube. By comparing the light that passes through the sample with
the same beam of light that bypasses the sample, the sample amount absorbed (“absor-
bance”) is determined. This measurement is both informative and straightforward. While
the method is only occasionally useful to identify functional groups in molecules (the
spectrum of light at different frequencies forms broad bands), it is very useful for quanti-
fication because the absorption is linearly related to concentration. The last statement is
formally known as “Beer’s Law”. With a standard curve of known concentrations of a mol-
ecule, a linear plot is invariably obtained, from which it is a simple matter to determine
the concentration of an unknown. For enzymatic reactions that produce (or consume) an
absorbing species, the absorbance can be measured as a function of time.
114 6.3 A Simple Kinetic Mechanism
FIGURE 6.4 Initial velocity. Product formation falls off with time. The greatest slope – and fast-
est rate – of this curve is near zero time; that is the asymptote shown as a dotted line. The slope
of this line is the initial velocity, the parameter used throughout enzyme kinetics.
complications of a continuously depleted substrate concentration, as well as effects of the
accumulated product. While the exact initial velocity strictly applies only as substrate con-
centration approaches zero, in practice, the first 10% of the reaction usually shows a nearly
linear slope.
Using the initial velocity measurements, as indicated in Figure 6.4, it is possible to charac-
terize enzymatic reactions by varying the substrate concentrations. To produce an equation
that can account for an enzymatic reaction, we need to suggest a plausible mechanism. It
happens that a very simple mechanism illustrates the key points and is broadly applicable to
the majority of enzymes.
6.3 A SIMPLE KINETIC MECHANISM
The simplest possible reaction is S → P, where S is the substrate, and P is the reaction prod-
uct. The enzymatic route consists of three steps: (1) the enzyme binds the substrate, (2) the
enzyme transforms the substrate into the product, and (3) the enzyme releases the product.
The entire process may be written as two chemical reactions:
(6.1) E + S ES
(6.2) ES ® E + P
In Equation (6.1), E and S reversibly combine to form the enzyme–substrate complex ES.
In Equation (6.2), the complex ES irreversibly breaks down to form P and regenerate E. The
net reaction – the sum of these two – is just S → P, the same as the nonenzymatic reaction.
Note that the enzyme assumes two forms over the course of the enzymatic reaction: the free
enzyme forms E and the bound enzyme forms ES.
We can also rewrite Equations (6.1) and (6.2) in a single line and assign rate constants for
each chemical step:
kf
(6.3)
E + S k cat
Es ¾¾® E + P
kr
The rate constants are proportionalities between substrate concentrations and reaction rate.
The rate constants in Equation (6.3) are:
k f: the forward rate constant for the formation of ES from E and S

kr: the reverse rate constant for the formation of E and S from ES
kcat: the rate constant for the breakdown of ES to E and P
The kcat rate constant, which leads to product formation, is also known as the catalytic rate
constant indicated by the subscript cat.
6.3.1 ASSUMPTIONS
Equation (6.3) is our model for enzyme kinetics. It is possible to derive an equation from this
model that relates the initial velocity v to the substrate concentration [S]. To accomplish this,
we make three assumptions:
1. Steady-State
The first assumption is that the system is in a steady-state. Both experimental
enzyme assays and pathways in living cells achieve a steady-state, at least when con-
sidered for longer than a fraction of a second. As ES is the only intermediate in the
process, the concentration of this species remains constant with time as the reac-
tion proceeds.
2. Enzyme Conservation
Second, we assume that the total amount of enzyme is unchanged throughout the
reaction. This assumption will always be true of the in vitro enzyme assay because
the total amount of enzyme, [E]tot, is fixed by the analyst. In vivo, the total amount
of enzyme is also constant over reasonably short periods, usually for hours to days.
It is subject to fluctuation at more extended periods as the enzyme protein may
be increased in production (protein synthesis) or decreased (protein degradation).
Here, we are concerned exclusively with the shorter time frame. Enzyme conserva-
tion is expressed as:
(6.4) [E]tot = [E] + [ES]

3. Initial Velocity is The Product Formation Step
Third, we assume that the initial velocity v can be identified as the step in the mech-
anism that leads to product formation. In our mechanism, this is the reaction gov-
erned by the catalytic rate constant (kcat):
(6.5) v = k cat [ ES]
6.3.2 THE MICHAELIS–MENTEN EQUATION
We can derive an equation that relates the initial velocity of an enzyme to its substrate con-
centration. The equation was named after its formulation by Leonor Michaelis and Maud
Menten, pioneers of enzymology (Box 6.3). All of the assumptions in the prior section are
applied to our enzyme mechanism (Equation 6.3), using the same principles for constructing
rate equations that we used in Chapter 1.
We start by expressing the steady-state assumption as:
(6.6) Rate of ES formation = Rate of ES destruction
We can cast this into expressions involving rate constants and concentrations taken from
our mechanism (Equation 6.3) and insert these on both sides of the equality of Equation (6.6)
to give:
(6.7) k f [ E][S] = k r [ ES] + k cat [ ES]

116 6.3 A Simple Kinetic Mechanism
Box 6.3 Historical Perspective of the Michaelis–Menten Equation
While these investigators are the most famous for devising the equation that applies to
most enzymes today, several others arrived at essentially the same formulation and con-
tributed fundamental concepts to the model, such as the notion of an enzyme–substrate
intermediate and the steady-state itself. These studies began in the late 19th century and
most focused on analyzing the enzyme invertase, named for its ability to alter the light
rotation of sucrose. The basis of this is that the reaction catalyzes the hydrolysis of sucrose
to glucose and fructose; the specific rotations of the reaction mixture changes sign as
products form. The values of the specific rotation (that is, light rotation corrected for the
concentration of a substance in g/ml at a set temperature) are (Table 6.3):
This analysis is not used widely in modern studies because other methods, such as
spectrophotometry, are simpler and more accurate. For the invertase (sucrase) reaction:
Sucrose ® glucose + fructose
there will be a net inversion, or change in sign in the instrument, because the specific
rotation of fructose is more negative than that of glucose; however, the latter will still
contribute and must be accounted for. The strength of the work of Michaelis and Menten
(beyond the derivation of the equation, which others also arrived at) was primarily their
experimental acumen in performing the analysis.
TABLE B6.3 Specific Rotation Values

Compound Specific Rotation
Sucrose +66
Glucose +53
Fructose −92
We can algebraically rearrange this equation, putting the concentrations on one side and the
rate constants on the other:

[E][S] = k cat + k r
(6.8)
[ES] kf
The collection of constants on the right side of Equation (6.8) is defined as K m (the Michaelis–
Menten constant):
k cat + k r
(6.9) Km =
kf
The second assumption is enzyme conservation. We start the reaction with a fixed, known
amount of enzyme and finish it with the same amount. The amount of enzyme is the amount
we add to the assay system, called the total enzyme concentration, or [E]tot. As the reaction takes
place, some of the enzyme is in the form of free enzyme E, and some is in the form of bound
enzyme ES. While these two can vary, their sum is constant; the conservation equation is:
(6.10) [E]tot = [E] + [ES]

This equation states that [E] and [ES] are not independent of one another as their sum is
constant. We can use this to replace the [E] term in Equation (6.8):

(6.11)
([ E ]
tot )
- [ ES] × [S]
= Km
[ES]
We now introduce our third assumption: the overall velocity of the reaction involves the step
leading to product formation. From our mechanism in Equation (6.3), this is the step involv-
ing the rate constant kcat:

(6.12) v = k cat [ ES]
To construct an expression for [ES] in terms of [S] and constants, we rearrange

Equation (6.11):
[ E ] [S]

(6.13) [ES] = K tot+ S
m [ ]
Substituting Equation (6.13) into Equation (6.12):
k cat [ E]tot [S]

(6.14) n=
K m +[S]
One last substitution is to introduce the definition:

(6.15) Vmax = k cat [ E]tot
We could also arrive at Equation (6.15) by considering Equation (6.12) when all of the enzyme
is in the ES form, so ES and Etot are the same. Substituting Equation (6.15) into Equation
(6.14) produces the Michaelis–Menten equation:
Vmax [S]
(6.16) v=
K m + [S]
The two constant terms that were defined in the course of deriving the Michaelis–Menten
Equation – Vmax and Km – are called kinetic constants. We will first examine how these
constants shape the velocity–substrate curve and then further explore their significance.
6.4 HOW THE MICHAELIS–MENTEN EQUATION

DESCRIBES ENZYME BEHAVIOR
A typical plot of initial velocity as a function of substrate concentration is shown in Figure 6.5.
The Michaelis–Menten Equation (Equation 6.16) correctly predicts the form of this dia-
gram. Note that the curve in Figure 6.5 looks the same as the equilibrium binding curve of
FIGURE 6.5 Michaelis–Menten plot. This graphs the initial velocity v versus substrate concen-
tration [S]. The curve approaches the asymptote Vmax at high [S] and the asymptote line with a
slope of Vmax /Km at low [S].
118 6.4 How the Michaelis–Menten Equation Describes Enzyme Behavior
Box 6.4 Mathematical Form and Scientific Interpretation
The distinction between the binding of oxygen to myoglobin and the velocity–substrate
curve for an enzyme is not apparent from their equations. In fact, they are mathemati-
cally identical. Each is in the form of what mathematicians describe as a rectangular hyper-
bola. The curve and its equation are the same for both proteins; it is only the mapping of
the terms that are different. Readers may wonder how they could be different. As a wise
author once said:
“Identical mathematical formulations must yet be viewed as different if they are interpreted
differently, especially if their purpose is scientific and not purely mathematical.”
from the introduction to The Rise of Statistical Thinking 1820–1900, T.M. Porter, Princeton
Press 1986
This notion is particularly important as we are contrasting two different models in these
equations, as stated in the text. It is critical to keep them separate as the equilibrium
model is not only the most familiar, but is often the default even when it is incorrect.
myoglobin to oxygen (Figure 5.28). However, the myoglobin-binding curve represents equi-
librium binding, and the Michaelis–Menten plot represents a steady-state, with a net flow of
material from substrate to product (see Box 6.4).
The curve of Figure 6.5 has two asymptotes drawn as straight dashed lines. These lines
extrapolate to the extremes of substrate concentration: infinity and zero. As [S] approaches
infinity (or in practical terms, becomes very large), v approaches the maximum velocity Vmax.
This asymptote is a horizontal line, and the value of Vmax corresponds to the intersection of
this line with the initial velocity axis. As [S] approaches zero, a second asymptote to the curve
is illustrated in Figure 6.5. This plot is a straight line that intersects at the origin and has a
slope of Vmax/Km. This slope is the ratio of the two constants that appear in the Michaelis–
Menten equation. Equations for both asymptotes can be derived by setting [S] to infinity or
zero and solving the Michaelis–Menten equation (Equation 6.16) for each situation.
For Vmax, we consider the limit of Equation (6.16) as S → ∞. That limit is:
Vmax [S] æ Vmax [S] ö

(6.17) v = lim =ç ÷ = Vmax
[ ] çè [S] ÷ø
[S]®¥ K m + S
because in the denominator, the [S] term overwhelms the Km. This equation explains why
the asymptote for very high (infinite) substrate concentration is horizontal. The velocity
becomes independent of [S]. It also provides a practical interpretation of what “infinite” con-
centration is:

(6.18) [S] >> Km
For the low substrate condition, we consider the limit of Equation (6.6) as S → 0. That limit is:
Vmax [S] Vmax [S]

(6.19) v = lim =
[ S ]® 0 K m + [ S ] Km
In this case, Km overwhelms [S] in the denominator, so only Km remains after taking the
limit. The velocity is now linearly related to [S]. Here too, we have a practical guide to deter-
mining how close to zero the [S] must be:

(6.20) [S] << Km
Unlike the kinetic constants Vmax and Km, their ratio Vmax/Km is a rate constant. That is appar-
ent from the form of Equation (6.19), in which this ratio is a constant that defines the propor-
tionality of the rate of the enzymatic reaction v to the substrate concentration [S].
The constants Vmax and Vmax/Km completely define the Michaelis–Menten equation, as
illustrated by Figure 6.5. Km is important, as it provides a reference point to determine how
high substrate concentrations must be as a practical matter to satisfy Equation (6.20). The Km
also serves as a reference point for low substrate concentrations for the asymptote at Vmax/
Km. There is a further significance of Km, which we explore next.
6.5 THE MEANING OF KM
The first point to appreciate about Km is that it has units of concentration because it is a con-
centration. In the last section, we saw that it could be used as a benchmark for high or low
substrate concentration in determining Vmax and Vmax/Km.
There is one value of [S] that corresponds to the midpoint of initial velocity, which is equal
to ½ Vmax. We can equate the initial velocity v to this value and solve the Michaelis–Menten
equation (Equation 6.16):
Vmax [S]
(6.21) v = ½Vmax =
K m +[S]
This reduces to:
(6.22) ½=
[S]
K m + [S]
This equation is satisfied when:
(6.23) K m =[ S ]
This equality is graphically demonstrated in Figure 6.6, which shows that the velocity point
at half of Vmax corresponds to the substrate concentration Km.
If we consider a substrate in its cellular environment, it is commonly found that the con-
centration of the substrate is very close to the value of Km. This correspondence is not sur-
prising because this is the region of concentration that provides the greatest capacity for an
increase or decrease in velocity as a function of [S]. Thus, the measured Km values provide an
estimate of intracellular concentrations.
FIGURE 6.6 The substrate concentration Km is reached by extrapolation to the corresponding

velocity equal to half of the Vmax.
120 6.6 Reversible Inhibition
There is another important use for Km. If an enzyme can use different substrates – such
as in the case of phosphatases – then their relative Km values can be used to differentiate
between them. Low values of Km indicate a tighter binding affinity to the enzyme. Caution
must be exerted in this application because Km is not an equilibrium constant, and affinity is
an equilibrium term. The actual affinity of a substrate, Ks, in terms of our model is:
kr
(6.24) Ks =
kf
Recall that:
k cat + k r
(6.9) Km =
kf
Comparing KS with Km, it is clear that they are only equal if kcat is equal to zero. That would
leave us with no product formation, and just the binding:
k
(6.25)
E + S
r

ES
k,
instead of the Michaelis–Menten model:
kf
(6.3)
E + S k cat
ES ¾¾® E + P
kr
Nonetheless, the assumption that Ks can approximate Km is frequently reasonable because

dissociation back to E and S usually proceeds far more rapidly than the formation of E and P
(that is, typically kcat << kr). The relatively low value of kcat is the assumption behind using an
equilibrium constant in place of the Km. It is useful for finding the approximate concentra-
tions of metabolites in cells, as well as for comparing different substrate affinities to enzymes.
However, there is a danger in overusing this assumption, which will become apparent in our
study of reversible inhibition.
6.6 REVERSIBLE INHIBITION
The most common means by which enzymes are regulated is through reversible inhibition.
Cellular regulators, as well as most drugs, are reversible inhibitors. Thus, it is vital to have a
clear understanding of how these agents work.
Reversible inhibitors bind to the active sites of enzymes (Box 6.5), and they are distin-
guished by their ability to bind different forms of an enzyme. By definition, a reversible inhib-
itor can establish an equilibrium with a form of the enzyme. In our simple model, there are
two forms available for binding to the inhibitor: E and ES. We can represent the interaction
between an inhibitor I and these forms by:

(6.26) EE+I K is = [ E][ I] / [ EI]
(6.27) ESI ES + I K ii = [ ES][ I] / [ ESI]
Where the labeling of the inhibition constants refers to a more general case of inhibition
described in Section 6.7.
Reversible inhibition can be categorized as competitive, anticompetitive, or mixed.
Competitive inhibition is defined as the binding of the reversible inhibitor (I) to the free
enzyme form only (Equation 6.26). Anticompetitive inhibition is defined as the binding of I
to the ES complex only (Equation 6.27). Mixed inhibition is defined as the binding of I both
E and ES forms (Equations 6.26 and 6.27).
Box 6.5 Inhibition and Active Sites
The active site of an enzyme is where the chemistry of catalytic events takes place.
However, it also includes the portion that binds directly to the substrates. Thus, the active
site is dynamic over the course of the reaction. While it might seem axiomatic to define
a competitive inhibitor as one that binds the active site and others as ones that do not,
this is incorrect. Competitive inhibitors bind the same enzyme form that the substrate
binds, but exactly where any type of reversible inhibitor binds is not revealed by a kinetic
analysis.
Commonly, we consider the binding of an inhibitor to a site other than the active site
to be an allosteric inhibitor. Indeed, this is the very definition of the term allosteric. It is
possible with current techniques to modify selected amino acids in enzyme active sites.
Such studies, followed by binding and kinetic analysis, help discriminate active sites from
allosteric ones.
For each type of inhibitor, we will establish three points. First, we will develop an intuitive
picture of how different types of inhibitors function. Then, we will determine how the model
for enzyme kinetics is modified by incorporating the equilibrium involving the inhibitor.
Finally, we will show how the inhibitor affects the velocity–substrate curve. For this purpose,
we need only determine effects at the extremes of very high and very low substrate concen-
trations, which will lead to how the inhibitor affects the two critical parameters: Vmax and
Vmax/Km.
6.6.1 COMPETITIVE INHIBITION
Competitive inhibitors often bear a structural similarity to the substrate. This chemical
resemblance stems from the fact that both the inhibitor and the substrate bind to the same
enzyme form, namely the free enzyme E. Conceptually, competitive inhibition is easy to pic-
ture because everyday examples are abundant. In ecology, organisms compete for food and
territory; in academics, students compete for grades; in finance, business people compete for
money.
Competitive inhibition can be modeled by adding the equilibrium reaction (Equation
6.26) to our standard model, producing:
E + S ES ® E + P
+

(6.28) I
↑↓ K i
EI
An equation to describe the interaction quantitatively can be derived as we have done for the
Michaelis–Menten equation, with the addition of a new equilibrium and an enzyme conser-
vation equation that includes the dead-end form EI (derived in Appendix A6). The resulting
equation is similar to the Michaelis–Menten equation, except that (1 + [I]/Kis) modifies the
Km term:
Vmax [S]

(6.29) v=
æ [ I] ö
Km ç 1 + ÷ + [S]
è K is ø
Figure 6.7 shows how a competitive inhibitor affects the velocity–substrate curve. As [S] →
∞, the inhibition disappears. The loss of inhibition can also be appreciated by examining
Vmax/Km
40
(Vmax/Km)’
Vmax
Control
v
20
+ competitive inhibitor
0 50 100 150 200

[S]
FIGURE 6.7 Competitive inhibition. A competitive inhibitor has its greatest influence at low
substrate concentration. There is a decreased value of Vmax /Km but no change in the value of Vmax.
how maintaining [I] constant and increasing [S] affects Equation 6.29. Since [I] is part of a
term that modifies the Km as [S] becomes very large, the equation becomes:
(6.30) v = Vmax
We conclude that a competitive inhibitor is ineffective at saturating substrate concentra-

tions. Competitive inhibitors provide a clear picture of why very high concentrations of sub-
strates are considered saturating, as we imagine they occupy all of the enzyme active sites
with substrate molecules, preventing binding of the inhibitor.
Competitive inhibition is most effective at very low substrate concentrations. In this case,
the modified Km term in the denominator of Equation 6.24 overwhelms the [S] term, as
[S] → 0. This limit is:
Vmax [S]
(6.31) v=
æ
Km ç 1 +
[ I] ö
÷
è K is ø
This equation shows analytically that the rate is still linear at low concentrations. In this
concentration condition, inhibition is maximal: an increase in [I] or a decrease in Kis (that is,
an increase in affinity, as Kis is a dissociation constant) will depress v. Our intuitive picture
of competitive inhibition is consistent, as more inhibitor molecules occupy the free enzyme
when the number of inhibitor molecules overwhelms the number of substrate molecules.
6.6.2 ANTICOMPETITIVE INHIBITION (UNCOMPETITIVE)
Anticompetitive inhibitors do not bind the free E form of the enzyme and generally do
not structurally resemble the substrate. Rather, an anticompetitive inhibitor binds to the
ES enzyme form. To gain an intuitive understanding, imagine a bear attempting to remove
honey from a hive with a narrow opening. The bear can move his empty paw in and out of
the opening with no difficulty. Yet, once his paw is filled with honey inside the hive, removal
is more difficult. Only the paw–honey complex is impaired; if the bear releases the honey, he
is not “inhibited”. Alternatively, consider a sting operation. A suspect is offered a lucrative
deal involving contraband. When she accepts, undercover agents arrest her. Only when she
associates with an illegal transaction is she at risk. Had she not become associated with it,
she would not be “inhibited”. In both cases, it is the bound form that is subject to inhibition.
We recall the interaction of an anticompetitive inhibitor with the enzyme form ES:
(6.27) ESI ES + I
and add this onto our standard mechanism (Equation 6.3), producing:
E + S ES ® E + P
+
(6.32) I
↑↓
ESI
The velocity–substrate equation for this model is:
Vmax [S]
(6.33) v=
æ [I] ö
K m +[S]ç 1 + ÷
è Ki ø
Figure 6.8 illustrates the influence of an anticompetitive inhibitor on the velocity–substrate

curve. In the region where [S] → 0, we observe no inhibition. The simplified equation for
velocity that prevails under this condition is Equation 6.34:
Vmax
(6.34) v= [S]
Km
with no inhibitor term remaining in the equation to affect Vmax/Km. We interpret this to
mean that unless the enzyme binds the substrate, no ES complex is formed. Because the
inhibitor only binds ES, with little to bind, there is little inhibition.
Inhibition appears strongest at the opposite extreme, where [S] → ∞. Under these condi-
tions, Equation 6.33 reduces to:
Vmax [S]
(6.35) v=
æ [I] ö
ç1 + ÷
è Ki ø
so that only Vmax is affected by the inhibitor.

Anticompetitive and competitive inhibitions are opposite extremes, which is why we use
the term “anticompetitive” instead of uncompetitive (Box 6.6). For a competitive inhibitor,
the more enzyme in the E form, the greater the inhibition. The more the enzyme is drawn
into the ES form for an anticompetitive inhibitor, the greater the inhibition.
40
Vmax/Km
Vmax
Control
20
v
Vmax’
+ anticompetitive inhibitor
0 50 100 150 200

[S]
FIGURE 6.8 Anticompetitive inhibition. An anticompetitive inhibitor has its greatest influence
at high substrate concentration. There is a decreased value of Vmax but no change in the value
of Vmax /Km.
Box 6.6 Word Origins: Uncompetitive and Noncompetitive
The established use of the words “uncompetitive” and “noncompetitive” has done much
to confuse generations of students – and investigators – as to the true nature of revers-
ible inhibition. For this reason, some specialists in enzyme kinetics have offered replace-
ments. We have used anticompetitive in place of uncompetitive because the action of
such inhibitors is the mirror image of competitive. Other names have also been pro-
posed, such as catalytic inhibition, because Vmax is decreased, and this kinetic constant
contains the rate constant kcat that leads to the product. The substitution of mixed for
noncompetitive evokes the correct picture of both a competitive (Vmax/Km effect) and an
anticompetitive (Vmax effect) inhibition. Others have suggested the term pure noncom-
petitive for the case in which both effects are equally inhibited, but this is merely a special
case of mixed inhibition. To underscore the confusion, a deaf student once pointed out
that American Sign Language does not discriminate between un- and non-. As the terms
are essentially equivalent, the words provide no clues to the types of inhibition, apart
from the fact that neither is competitive. Since they remain in wide use, the terms are
included here as parenthetic nomenclature, taking second place to the terms that assist
our understanding of the inhibition types.
6.6.3 MIXED INHIBITION (NONCOMPETITIVE)
A mixed inhibitor binds both to the free enzyme form E and to the bound enzyme form
ES. In essence, it is not a new form of inhibition at all but merely a combination of competi-
tive and anticompetitive inhibition. There are thus two equilibria to be added to the kinetic
model:
E + S ⇌ ES → E + P
+ +
(6.36) I I
⇅ Kis ⇅ Kii
EI ESI
We will consider first the situation in which the values of the constants K is and Kii are identi-
cal. At low [S], inhibition will result because I can bind the E form. Accordingly, we expect
to observe a diminished Vmax/Km, just as in the case of the competitive inhibitor. At high [S],
inhibition will result because the inhibitor can bind the ES form. Accordingly, we expect
to observe a diminished Vmax, just as in the anticompetitive inhibitor case. At any substrate
concentration in between the very low and very high, the mixed inhibitor will also reduce v,
as shown by the following rate equation (see Appendix A5 for the derivation):
Vmax [S]

(6.37) v=
æ [I] ö æ [I] ö
K m ç 1 + ÷ + [S]ç 1 + ÷
è Ki ø è Ki ø
and by the graph of Figure 6.9. Mixed inhibition with a single value for K i can be visualized
as just removing some of the enzyme, because forms E and ES are equally diminished by the
inhibitor, and [E]tot is the sum of these forms.
In the more general situation, the Kis and Kii values are not equal. We can make a first
approximation to this inhibition behavior by realizing that the equilibrium constant rep-
resenting the strongest binding will be dominant. For example, if I binds preferentially to
E (i.e., Kis < Kii), the inhibition will resemble competitive inhibition. On the other hand, if I
40
Vmax/Km
Vmax
Control
20
v
+ mixed inhibitor
0
0 50 100 150 200
[S]
FIGURE 6.9 Mixed inhibition. A mixed inhibitor decreases both Vmax and Vmax /Km. The situa-
tion shown here has both inhibitor binding constants the same, so that inhibition is the same at
low and high substrate concentrations – and thus the same inhibition is exerted at all substrate
concentrations.
FIGURE 6.10 The double–reciprocal plot. A straight line results from plotting a rearranged
Michaelis–Menten equation so that 1/v is expressed as a function of 1/[S]. The indicated intersec-
tions provide estimates of Vmax and Km.
binds preferentially to ES (Kis > Kii), the inhibition will resemble anticompetitive inhibition.

In either case, there will be a slight inhibition for the weaker binding species, rather than the
complete lack of inhibition that occurs in the competitive or anticompetitive cases. Another
view of reversible inhibition is that mixed inhibition is all-encompassing, with competitive
and anticompetitive inhibition as special cases in which binding to E or ES dominates.
6.7 DOUBLE-RECIPROCAL OR LINEWEAVER–BURK PLOT
The hyperbolic Michaelis–Menten curve can be transformed into a straight line by rearranging
Equation 6.6 and plotting 1/v versus 1/[S]. This graph is known as the double–reciprocal or
Lineweaver–Burk plot. The values of Vmax and Km appear at the intersections of its axes (Figure
6.10). Note that Vmax/Km, not Km, is required for interpreting enzyme inhibition (Box 6.7).
In research studies, double–reciprocal plots are not used to determine kinetic constants
because the process of forming reciprocals emphasizes the smallest values, which are the least
experimentally reliable. The problem is that the smallest values become the largest values in
126 6.8 Allosteric Enzymes
FIGURE 6.11 Patterns of reversible enzyme inhibition. The three types of inhibition are graph-
ically distinct when plotted in double–reciprocal form. In each case, increasing the amount of
inhibitor produces new lines showing alterations in the slope (competitive), intercept on the 1/
[S] axis (anticompetitive), or both (mixed).
the reciprocal and receive the most significant statistical weight in extrapolating the straight
lines. However, reciprocal plots are widely used in inhibition studies because each type of
inhibitor produces a distinct pattern. Constructing double–reciprocal plots with various
amounts of inhibitor present produces lines that intersect at the 1/v axis (competitive inhibi-
tion) are parallel (anticompetitive inhibition) or intersect to the left of the 1/v axis (mixed
inhibition) as shown in Figure 6.11. The naming of the two dissociation constants for binding
to enzyme forms is based on the appearance of these curves. Thus K is for binding to the free
enzyme indicates a slope effect, which is the pattern of competitive inhibition. K ii for binding
of the bound enzyme shows an intercept effect, the signature of anticompetitive inhibition.
Both slope and intercept effects are apparent in the case of mixed inhibition.
6.8 ALLOSTERIC ENZYMES
Not all enzymes display the velocity–substrate behavior that we have examined so far. One
well-known deviation is that displayed by allosteric enzymes. The term allosteric (Greek for
FIGURE 6.12 Allosteric enzymes and cooperative behavior. Allosteric enzymes usually display
cooperative behavior, in which substrate binding alters the ability of the enzyme to bind further
substrate molecules, reflected in a steadily increasing velocity. Eventually, the enzyme is satu-
rated, and velocity falls off to Vmax, as with all enzymes.
FIGURE 6.13 Inhibitors and activators of allosteric enzymes. Modulators of allosteric enzymes
cause a shift to the right (inhibitors) or left (activators), greatly extending the influence of these
compounds on enzyme velocity over Michaelis–Menten type enzymes.
other place) indicates that modifiers of the enzyme bind to a place other than the active site at
which substrates are converted to products. Their ability to influence the active site remotely,
as it were, stems from conformational changes in the protein typically transmitted between
separate subunits of a protein.
Allosteric enzymes are usually composed of multiple subunits that interact during cataly-
sis. The model shown in Figure 6.12 is one possible mechanism. Substrate binding alters
subunit conformations, indicated in the figure as a transformation from spheres to blocks.
The blocks represent an increased catalytic ability for the subunit. This behavior is called
positive cooperativity, an idea borrowed from the equilibrium hemoglobin-oxygen bind-
ing curve (Chapter 5). Inhibitors of cooperative enzymes shift the curve to the right because
they decrease the relative effectiveness of the substrate on v, as indicated in Figure 6.13. Also
shown in Figure 6.13 is the shift of the curve to the left, the response to an allosteric activator.
In a similar way, covalent modifications of enzymes can lead to alterations in v. The most
common one is the incorporation of a phosphoryl group from ATP onto the side chain
hydroxyl group of a serine, threonine, or tyrosine side chain. The phosphorylated enzyme may
128 6.9 Irreversible Inhibition
display allosteric activation or inhibition identical to the kinetic curves shown in Figure 6.13.
Restoring the enzyme to its non-phosphorylated form requires a protein phosphatase, which
also returns the enzyme to its original activity. While the effects of phosphorylation on
enzyme activity are similar to allosteric regulators, there is a distinction. Once an enzyme is
covalently modified, it can no longer be considered reversibly inhibited (or activated).
6.9 IRREVERSIBLE INHIBITION
Covalent modification of an enzyme that leads to a decrease in its activity is irreversible

inhibition. As the modifying agent is covalently attached to the enzyme, there is no equilib-
rium. The phosphorylation example presented in the prior section is an example of a physi-
ologically irreversible modulation. A toxicological example is the reaction of the nerve gas
diisopropylfluorophosphate with acetylcholine esterase, as shown in Figure 6.14. Once modi-
fied, this enzyme activity cannot be restored.
Normally, acetylcholine esterase catalyzes the hydrolysis of the ester bond in acetylcho-
line, producing the inactive molecules acetate and choline (Figure 6.15). Acetylcholine is the
neurotransmitter that signals muscle contraction from the connecting nerve; if it cannot be
hydrolyzed, the signal remains high, muscles go into uncontrolled contraction (called con-
tracture), and death ensues rapidly, as muscle relaxation is essential for breathing.
There are several examples of drugs that act as irreversible inhibitors, including aspirin
(which inhibits prostaglandin and prostacyclin formation), penicillin (which disrupts the for-
mation of bacterial cell walls), and Prilosec (which inhibits the proton pump in the stomach,
thereby reducing the amount of stomach acid).
FIGURE 6.14 Irreversible inactivation of acetylcholine esterase. The enzyme is covalently

modified by diisopropylfluorophosphate, which has a phosphoryl nucleus subject to attack
by the serine hydroxyl of the enzyme, and an attached fluoride, a good leaving group. Once
formed, the modified enzyme is no longer reactive towards its substrate.
FIGURE 6.15 Mechanism of acetylcholine esterase. The serine residue of the enzyme attacks
the ester of the substrate acetylcholine and forms an enzyme intermediate with choline release.
Subsequently, this adduct is hydrolyzed, the free form of the enzyme is reformed, and the sec-
ond substrate, acetate, is released.
6.10 ENZYME MECHANISMS
In our study of enzyme kinetics so far, we have suppressed the molecular nature of the reac-
tants, using the symbols E, S, ES, etc. A mechanism for the enzyme acetylcholine esterase is
outlined in Figure 6.15. The alcohol of an enzymatic serine side chain reacts with the carbonyl
of acetylcholine, with electron rearrangements as shown to produce an enzyme-bound inter-
mediate, an acetylated serine residue, releasing choline. Subsequent ester hydrolysis releases
acetate and regenerates the free enzyme. The mechanism is cast into the familiar symbols
used in enzyme kinetics, with the assignments shown in Figure 6.16a and the mechanism in
symbolic form in Figure 6.16b.
The chemistry of enzyme mechanisms is a topic that we explore throughout this text.
Using the example of acetylcholine esterase, we can demonstrate two of the most common
types: nucleophilic substitution and acid–base catalysis.
6.10.1 NUCLEOPHILIC SUBSTITUTION
Many reactions in biochemistry are nucleophilic substitutions. In the case of acetylcholin-

esterase, the key step is the attack of the oxygen of the hydroxyl group (which is relatively
electron-rich) on the carbonyl carbon (which is relatively electron-poor). As indicated in
Figure 6.17, the electron pair from the serine oxygen attaching to the carbonyl carbon is fol-
lowed by migration of a pair of electrons from the double bond of the carbonyl to the oxygen
atom. In the second line of the mechanism, it is evident that the extra electrons now on the
130 6.10 Enzyme Mechanisms
FIGURE 6.16 Symbolic form of the acetylcholine esterase mechanism. (a) The symbols used
in enzyme kinetics correspond to the mechanism of acetylcholine esterase. (b) A symbolic
mechanism.
FIGURE 6.17 Nucleophilic substitution in acetylcholine esterase. Details of the nucleophilic

substitution are shown, a common mechanism in enzyme chemistry. The migration of electrons
is indicated as curved arrows. An intermediate is formed first; subsequent electron migration
leads to rupture of the C–O bond of the original ester.
oxygen atom return to the double bond of the carbonyl; a final migration of electrons splits
the C–O bond and produces the acetylated enzyme. As outlined in Figure 6.15, hydrolysis of
the acetylated enzyme regenerates the free enzyme and releases acetate.
6.10.2 ACID–BASE CATALYSIS
Often a group is made more reactive by an acid–base reaction. At the simplest level, we can
think of this as adding or removing a proton. In rare cases, the entire mechanism is just acid–
base catalysis. It is usually a part of another mechanism, and the proton donor or acceptor
is an amino acid residue of the enzyme. In acetylcholine esterase, a histidyl residue acts as
a base to remove a proton from the serine hydroxyl group during the nucleophilic attack by
the oxygen. Thus, acid–base catalysis is part of a concerted mechanism for the nucleophilic
substitution (Figure 6.18).
Notice how the base of histidine is itself assisted by a neighboring glutamate residue
shown in the figure. The relay of electron pair movements shown in Figure 6.18a is an assist
to the acid–base mechanism. This arrangement of three amino acids performing acid–base
assistance to a nucleophilic attack is known as a catalytic triad. There are many other exam-
ples of catalytic triads in enzyme mechanisms, often with the same or similar amino acids
participating.
FIGURE 6.18 The catalytic triad. Three amino acid residues of acetylcholine esterase are
shown positioned close together. They undergo concerted acid–base reactions that enable pro-
ton extraction from the serine hydroxyl group coincident with oxygen atom – from the same
hydroxyl group – attacking the carbonyl of the substrate.
132 6.11 Enzyme Categories
TABLE 6.1 Classification of Enzymes by the

First Category of the Enzyme Commission (EC)
EC Type Category Example
1 Oxidoreductase Lactate Dehydrogenase
(Chapter 9)
2 Transferase Hexokinase
(Chapter 9)
3 Hydrolase Sucrase
(this chapter)
4 Lyase Aldolase
(Chapter 9)
5 Isomerase Glucose Phosphate Isomerase
(Chapter 9)
6 Ligase Pyruvate Carboxylase
(Chapter 13)
7 Translocase Cytochrome c Oxidase
(chapter 10)
6.11 ENZYME CATEGORIES
While we have examined the details of one enzymatic reaction, there are thousands more.
Fortunately, there are far fewer reaction types. The categorization of the Enzyme Commission,
a committee of biochemists, places enzymes into groups and subgroups. We will consider
the top level of this hierarchy, which will provide a broad description of what is taking place
between substrate and product. There are just seven categories (Table 6.1).
The first types, the oxidoreductases, are also called dehydrogenases or reductases.
These enzymes catalyze changes in the oxidation state. More enzymes are oxidoreductases
than any other class.
Transferase enzymes catalyze reactions that move a piece of one substrate onto another.
This category is also common and includes all examples in which transfers involve the high-
energy compound ATP, such as hexokinase in Table 6.1. In a broad sense, it is also the cat-
egory of reaction for the schematic reaction of Figure 1.1.
The hydrolase enzymes are formally subsets of the transferases, in which water is a sub-
strate. Enzymes located in the exterior of cells such as lactase (Table 6.1) are often hydrolases
because cofactors (other than water) are unnecessary. As a further example, all digestive
enzymes secreted into the intestine – such as ribonuclease – are hydrolases.
Lyase enzymes catalyze the splitting of a substrate into pieces. This may seem superfi-
cially similar to hydrolases, but lyase reactions do not involve water as a substrate.
The class of isomerase enzymes catalyze rearrangements, such as shifting the location of
a hydroxyl group. This alteration often changes the molecule’s stereochemistry, as in the case
of the enzymes racemase and epimerase.
Ligase enzymes catalyze joining reactions that also involve a high-energy donor (usually
ATP). Ligases are sometimes called synthetases. The very similar term synthase is a syn-
onym for a lyase (EC 4), with the reaction written in the opposite direction.
The last category, translocase, is also the newest, added in 2018. These are enzymes that
catalyze transport across a cellular membrane as well as a chemical transformation. The
example listed in Table 6.1 is part of the mitochondrial respiratory chain. It oxidizes cyto-
chrome c, transforms oxygen into water, and transports protons from the inside to the out-
side of mitochondria.
6.12 ENZYME-LIKE QUALITIES OF MEMBRANE TRANSPORT PROTEINS
Many of the proteins embedded in cell membranes enable the selective exchange of small
molecules between biological water spaces. These are transporters, each of which is selective
FIGURE 6.19 Transporters as enzymes. The ability of transporters to “catalyze” the movement
of substances from one biological space to another is directly analogous to enzyme action. The
reaction is sketched in (a), and in symbolic form in (b).
for a specific molecule or class of molecules to cross a membrane. Some of these are now
classified as translocase enzymes (EC 7), but only if they also involve a chemical transforma-
tion. Those proteins that perform a transport function exclusively fall outside of the enzyme
classification but share features of enzymes.
For example, the glucose transporter (Figure 6.19) allows extracellular glucose to cross
the plasma membrane of most cells and enter the cytosol. If we take v as the rate of glucose
entry into the cell interior, a plot of v versus glucose concentration shows the same form as
the Michaelis–Menten plot of Figure 6.5. Substituting extracellular glucose for S and intra-
cellular glucose for P, we have a “reaction” in which the transporter is catalyzing a transloca-
tion, or change in location, for the glucose molecule in the same way as an enzyme catalyzes
a chemical event. Even the ES complex in the enzyme mechanism is mirrored as a similar
enzyme-transporter complex. As we learn more about enzymes and their regulation, we will
find that transporters share these features. Thus, we should consider transporters – even
without a chemical transformation – as another form of catalysis, in which a change in space
rather than a change in chemical composition occurs.
SUMMARY
Enzymes are measured by the reactions they catalyze. The study of the rates of conver-
sion of substrates to products is called kinetics. The fundamental nature of catalysis is
to lower the energy barrier required for reactions to proceed, called the energy of activa-
tion. Enzymes lower the energy of activation by providing binding sites for substrates and
positioning reactive groups. The rates at which enzymes catalyze reactions are measured
using initial velocity v, the beginning of the time course before appreciable substrate has
been depleted or product has accumulated. In contrast to thermodynamics, kinetic studies
depend upon a pathway or model to convert substrates to products. The Michaelis–Menten
model envisions a substrate forming an intermediate complex with the enzyme – the ES
complex – and then releasing the product. A key assumption in developing an equation
that describes velocity as a function of the substrate is that of the steady-state, that is, the
rate of formation of an ES intermediate is equal to the rate of its destruction. The resulting
Michaelis–Menten equation has two constants, K m and Vmax. Km is a substrate at which the
initial velocity is half-maximal. Vmax is an initial velocity that occurs when the enzyme is
saturated with substrate. The ratio Vmax/Km characterizes the Michaelis–Menten equation
Box 6.7 Can Km Explain Enzyme Inhibition?
In a word, no! It is popularly thought that Km is a useful constant in enzyme inhibition

because it seemingly gives the “right” answer when applied to competitive inhibition. In
some circumstances, we have seen that Km can be approximated by Ks, the equilibrium
constant of ES dissociation. If we can continue to make this assumption for enzyme inhi-
bition, then Km is roughly an affinity constant for substrate binding (lower values mean
tighter binding). Table B6.7 shows that a competitive inhibitor increases the measured
Km. The temptation to conclude that a competitive inhibitor decreases the affinity of the
enzyme for the substrate is difficult to resist.
The problem with giving in to this temptation is evident by the rest of the Table. For
example, the anticompetitive inhibitor causes a decrease in Km. If we followed the same
path, we would have to conclude that this form of inhibitor increases the binding of
substrate to enzyme! Finally, for mixed (noncompetitive) inhibitors, when both binding
constants are the same, there is no change in Km. We would have to conclude that the
substrate affinity was unaffected by the presence of a mixed inhibitor. The interpretation
of inhibitory behavior based on assuming Km is an equilibrium constant is inappropriate.
The reason for the error is that Km is not really an equilibrium constant. At times, mak-
ing the approximation is reasonable, but making this assumption requires that v = 0.
Inhibition analysis is intrinsically a steady-state rather than an equilibrium process. In the
Table B6.7, alterations in the true kinetic constants, Vmax, and Vmax/Km, are regular and sym-
metric with the different inhibition types.
TABLE B6.7 Influence of Reversible Inhibitors on Vmax, Vmax/Km, and Km

Inhibition type Common Name Vmax Vmax/Km Km
Competitive Competitive – ↓ ↑
Anticompetitive Uncompetitive ↓ – ↓
Mixed Noncompetitive ↓ ↓ –
at low substrate concentration. This ratio is the rate constant for the enzymatic reaction
when [S] << Km. Reversible enzyme inhibition can be readily understood by considering the
extremes of substrate concentration. The key parameters are Vmax and Vmax/Km. Inhibitors
that primarily affect the enzyme at very low substrate concentration (i.e., decrease Vmax/Km)
are competitive; those affecting the enzyme primarily at very high substrate concentration
(i.e., decrease Vmax) are anticompetitive; those that affect both Vmax and Vmax/Km (and hence
all substrate concentrations) are mixed. Some inhibitors are not reversible; they covalently
bind to the enzyme and inactivate it permanently. Not all enzymes display the behavior
of the Michaelis–Menten model. Another important model is that of allosteric enzymes.
Allosteric enzymes have a sharper rise in their velocity–substrate curves, and modulators
shift the curve to the right (inhibitors) or left (activators). Allosteric behavior is the result
of subunit interactions following substrate binding. The chemical events of enzymes – the
detailed mechanism – often uses acid–base catalysis or nucleophilic substitution. Both
are involved in the catalytic triad mechanism of acetylcholine esterase. Enzymes may be
categorized as oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases, or
translocases. Finally, membrane transport proteins that do not perform chemical transfor-
mation are directly analogous to enzymes in that they catalyze the movement of a molecule
between different biological spaces.
REVIEW QUESTIONS
1. The Km is a collection of rate constants. For our simple mechanism, how would you
state in general the conditions under which the K m is approximately the same as the
dissociation constant? What is the fundamental difference between Km and a disso-

ciation constant (or the reciprocal, an affinity constant)?
2. It is very common to observe that the concentration of compounds within cells is
very close to the Km values of enzymes that utilize these same compounds as reac-
tants. Why do you think this is true?
3. Why isn’t Vmax the same for a given enzyme under all conditions?
4. What are the units of the constants: k f, kcat, kr, Km, Vmax, Vmax/Km?
5. The Eact (energy of activation) is a single peak of energy for an uncatalyzed reaction,
but at least two peaks in the enzyme-catalyzed reaction are shown for our model.
Why must this be the case?
6. What forms of the enzyme are bound by a competitive inhibitor? An anticompetitive
inhibitor? A mixed inhibitor? An irreversible inhibitor?
7. Suppose a more realistic model of enzyme kinetics was derived, with several interme-
diates. How would you modify your answer to the previous question?
8. If a reaction is at equilibrium, what would be the effect of adding more enzyme?
9. When cells are induced to alter their state of protein synthesis, the total amount of
enzyme is changed. Suppose this change leads to an increase in the amount of the
enzyme by a factor of 3. How will this affect the Vmax? The Km? The Vmax/Km?
10. Drugs are often enzyme inhibitors. Researchers interested in drug design find it easi-
est to construct a competitive inhibitor, but they are not the most desirable as drugs.
What do you think the explanation of this might be?
11. What is a rate constant, exactly? Is there any difference between a rate constant used
to express a non-enzymatic reaction and a rate constant used in enzyme kinetics?
12. In the derivations in this chapter, a steady-state assumption was used to describe
the interaction of enzyme with substrate, but an equilibrium was used to describe
the interaction of enzyme with inhibitors. Why couldn’t a steady-state assumption
also be used for inhibitors? Why couldn’t an equilibrium assumption be used for
substrates?
13. Allosteric enzymes do not follow the Michaelis–Menten equation, yet the steady-
state assumptions, enzyme conservation, and formation of enzyme–substrate com-
plex still apply. What feature makes allosteric enzymes unusual?
14. The form of the Michaelis–Menten equation and the form of the binding equilibrium
of myoglobin are similar. Not only that, the form of the allosteric enzyme curve and
the hemoglobin curves are also similar. Discuss the reason for the similarity and how
these are different.
15. What types of membrane transport proteins are not classified as EC 7?
16. Why is histidine the amino acid residue used as the centerpiece of the catalytic triad?
Could another amino acid be substituted for it?
CHAPTER 6 ADDENDUM: THE HALDANE RELATIONSHIP
When we measure enzyme activity, we always use the initial velocity and assume that no
product is present. If we relax the last condition, we have a reversible mechanism:
k1 k2

(A6.1)
E+S
ES
+ P
k -1 k -2
with the indicated rate constants. To arrive at a rate equation for this model, we must include
flows through the k-2 step, which modifies our steady-state assumption. When this is done,
the derivation leads to two Km values, one for the substrate and one for the product:
k -1 +k 2

(A6.2) K mS =
k1
k -1 +k 2

(A6.3) K mP =
k -2
136 Chapter 6 Addendum: The Haldane Relationship
We will also need two expressions for the product-forming step. The forward direction is the
same as before, except that rather than kcat the rate constant is named k 2:

(A6.4) n f = k 2 [ ES]
There is also a rate for the reverse direction:

(A6.5) v r = k -1 [ ES]
The enzyme conservation equation is the same as before:

(A6.6) [E]tot = [E] + [ES]
The two Vmax values are therefore:

(A6.7) Vmax(f) = k 2 [ E]tot

(A6.8) Vmax(r) = k -1 [ E]tot
The net rate of reaction in the forward direction is:
Vmax ( f ) [S] Vmax (r ) [P ]

-
K mS K mP

(A6.9) n=
1+
[ ] + [P ]
S
K mS K mP
From this equation, we can account for the presence of P, which will reduce the forward veloc-
ity. (As a check, notice that setting [P] = 0 reverts the expression to the simpler Michaelis–
Menten equation we have derived in this chapter). We can apply the equilibrium condition,
v = 0 and obtain an expression for the equilibrium constant reminiscent of Chapter 1. In this
case, the numerator of Equation A6.9 will also equal zero, and the concentrations of S and P
will be their equilibrium values:

(A6.10) v= - = 0
K mS K mP

(A6.11) =
K mS K mP

Vmax ( f )K mP
=
[P ] = K
(A6.12)
[S]
eq
Vmax (r )K mS
Equation A6.9 is known as the Haldane relationship, in honor of the biochemist J.B.S.
Haldane who first derived it. It demonstrates a relationship between kinetic constants and
the equilibrium constant. It shows that the Vmax and Km values for forward and reverse direc-
tions for enzymatic reactions are constrained in a similar way to the forward and reverse rate
constants for a nonenzymatic equilibrium reaction.
If we rearrange the Haldane relationship to collect the kinetic constants associated with
each direction, we can express Equation A6.9 as:
Vmax ( f )
K mS
(A6.13) = K eq
Vmax (r )
K mP
The ratio of Vmax/Km itself is the first order rate constant for v (or the slope of the asymptote
when [S] → 0). Thus, the ratio of these rate constants is also equal to the reaction equilibrium
constant.
KEY TERMS
activation energy
active site
affinity
allosteric enzymes
assay
catalytic rate constant
catalytic triad
competitive inhibition
concerted
dehydrogenases
double–reciprocal
Haldane
hydrolase
initial velocity
irreversible inhibition
isomerase
kinetic constants
Km
ligase
Lineweaver–Burk plot
lyase
mixed inhibition
oxidoreductase
positive cooperativity
reductases
synthase
transferase
translocase
transporter
uncompetitive inhibition
Vmax
Vmax/Km
BIBLIOGRAPHY
C.D. Berdanier, L. Berdanier. Advanced Nutrition: Macronutrients, Micronutrients, and Metabolism.
CRC Press. 2015.
P.F. Cook, W.W. Cleland. Enzyme Kinetics and Mechanism. Garland Science, London; New York. 2007.
A. Cornish-Bowden. Fundamentals of Enzyme Kinetics. 3rd ed. Portland Press, London. 2004.
G. Dodson, A. Wlodawer. Catalytic Triads and Their Relatives. Trends Biochem. Sci. 23 (1998) 347–352.
F.L. Lambert. Why Don't Things Go Wrong More Often? Activation Energies: Maxwell's Angels,
Obstacles to Murphy's Law. J. Chem. Educ. 74 (1997) 947.
R.S. Ochs. Understanding Enzyme Inhibition. J. Chem. Educ. 77 (2000) 1453–1456.
R.S. Ochs. The Problem with Double Reciprocal Plots. Curr. Enzyme Inhib. 6 (2010) 164–169. The
errors incurred with double reciprocal plots are discussed in detail. There are further comments
in the appendix.
R.S. Ochs, C.R. Ashby, Jr. Viewpoint: Discriminating between Noncompetitive and Allosteric
Interactions. Synapse 62 (2008) 233–235. DA - 20080108IS - 0887-4476 (Print)IS - 0887–4476
(Linking)LA - engPT - Journal ArticleSB – IM.
138 Bibliography
I.H. Segel. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme
Systems. Wiley-Interscience, New York. 1975.
K. Tipton. Translocases (Ec 7): A New Ec Class. 2018.
W.C. Vosburgh. The Optical Rotation of Mixtures of Sucrose, Glucose and Fructose. J. Am. Chem.
Soc. 43 (1921) 219–232. Analysis of the specific rotations of the sucrase catalyzed reaction for
experimentalists of the 1920's.
J. Wang, J. Gu, J. Leszczynski. Theoretical Modeling Study for the Phosphonylation Mechanisms of the
Catalytic Triad of Acetylcholinesterase by Sarin. J. Phys. Chem. B 112 (2008) 3485–3494.
Coenzymes 7
A coenzyme is a small molecule that associates with an enzyme, expanding its chemical rep-
ertoire. By small, we mean a molecule with no higher-order structure (i.e., unlike a protein or
polysaccharide polymer); typically, coenzymes have a molecular weight on the order of 1000.
The need for more elaborate chemistry in enzyme catalysis will become clear as we explore
the commonly encountered coenzymes.
Many enzymes can catalyze reactions exclusively using their aminoacyl functional
groups, such as acetylcholine esterase in the previous chapter. Aside from new chemical
possibilities, there is a second reason enzymes utilize coenzymes. In some cases, electrons or
a phosphoryl group must be transferred between enzymes within a cell space. We call such
carriers mobile coenzymes.
7.1 COENZYMES: BOUND AND MOBILE
We can divide coenzymes into two types: bound (prosthetic groups), and mobile. Bound
coenzymes function as part of the enzyme itself, attached by covalent bonds or strong non-
covalent forces so that they are not detached from the enzyme over many catalytic cycles.
While a bound coenzyme can undergo chemical change within an enzymatic cycle, it must
regain its original chemical form at the end of the reaction, just as the rest of the enzyme
does. Mobile coenzymes (or mobile cofactors) do not regain their original chemical form at
the end of the reaction. From the standpoint of an individual reaction, a mobile cofactor is
indistinguishable from a substrate or product. To underscore this point, they are also known
as cosubstrates and coproducts. Separate enzymatic reactions must regenerate the original
form of mobile cofactors.
We have already encountered a prosthetic group: the heme of myoglobin and hemoglobin
(Chapter 5). In the latter proteins, heme enables oxygen binding, which is not possible using
only amino acid side chains. Prosthetic groups that occur in enzymes are bound cofactors,
needed in small quantities – only as much as the enzymes themselves – and assist the chemi-
cal mechanism of catalysis.
The most prevalent mobile coenzyme is ATP, described in Chapter 4, and introduced as a
reactant in forming phosphorylated enzymes in the previous chapter. The protein modifica-
tion can be summarized as:

(7.1) ATP + Prot ® ADP + Prot - PO3 2 -
where ATP is the cosubstrate and ADP is the coproduct; Prot and Prot–PO32− represent the
protein and phosphorylated protein respectively. Continuous regeneration of mobile cofac-
tors is needed because the amounts of ATP and ADP are limited in cells, just like all coen-
zymes. If external reactions did not turn them over, they would be metabolic intermediates
and would need to be continually supplied and removed. While ATP is the most prevalent
nucleotide, others are also used in some reactions, such as GTP and CTP. Additionally, the
dephosphorylated nucleotides, as well as inorganic phosphate itself, must also be considered
mobile coenzymes. We will further consider nucleotides and inorganic phosphate in later
139
140 7.2 Coenzymes and Vitamins
chapters; they are involved in hundreds of biochemical reactions. In the bulk of this chapter,
we will consider the other major coenzymes.
7.2 COENZYMES AND VITAMINS
The majority of the coenzymes are derived from vitamins, which are substances that organ-
isms require in their diet as they do not synthesize them. In extreme cases, exclusion of a
vitamin from the diet results in an overt disease state. Consider the case of thiamine, the first
vitamin discovered. In Asian populations, a diet consisting mainly of polished rice led to out-
breaks of beriberi, with muscle weakness and swelling among its symptoms. The discarded
rice husks are a rich source of thiamine, which was chemically identified as a “vital amine”,
and, hence, the portmanteau word vitamin was born.
While it was once believed that all vitamins would be amines, it is clear that they are
structurally diverse (Table 7.1). Most vitamins were assigned the letter B with a number (e.g.,
B1, B2) to distinguish new members of the “vitamin B complex”. The only common feature
of this group – which accounts for most of the vitamins – is that they are water-soluble. A
major lipid-soluble vitamin that is a coenzyme is Vitamin K or phylloquinone. Menadione
serves the same function as phylloquinine, and the two are structurally related, with identi-
cal fused ring structures but a different length of a repeated branched-chain lipid. Menadione
is synthesized by our gut bacteria and meets about half the requirement for this coenzyme.
Table 7.1 lists one form of the most common vitamins, yet alternative structures exist in
nature that can be metabolically converted into the active coenzyme in humans. For exam-
ple, there are three forms of pyridoxine: alcohol, aldehyde, and amine. All are converted into
a single coenzyme form.
The major coenzymes, including both vitamin-derived and endogenously synthesized,
can be organized into four groups: redox, acyl transfer, carboxylation, and exchange. We will
consider the essential reactions in this chapter and expand on how the coenzymes are used
in the context of enzymes and metabolic pathways in ensuing chapters.
7.3 REDOX COENZYMES
Oxidoreductase is the largest Enzyme Commission category, accounting for about half of all
known enzymes. Three types of redox coenzymes are shown in Table 7.2: nicotinamides, qui-
nones, and flavins. The nicotinamides transfer hydrides, which are hydrogen nuclei attached
to a pair of electrons. The quinones – ubiquinone (UQ) and plastoquinone (PQ) transfer
electrons strictly one at a time. The flavin nucleotides are bifunctional. They can carry out
one-electron transport in one direction and two-electron transport (i.e., a hydride) in the
other. Flavins can serve as coupling redox carriers between strict two-electron coenzymes
(e.g., NADH) and single electron coenzymes (e.g., UQ).
7.3.1 NICOTINAMIDES
The nicotinamide nucleotides have both a niacin (nicotinamide) ring and an adenine ring
(Figure 7.1). As with all redox cofactors, there must be an oxidized and reduced partner on
each side of the reaction. There are two pairs of nicotinamide nucleotides: NAD+/NADH and
NADP+/NADPH. The full structure illustrated at the top of the figure indicates the nucleo-
tide rings in gold and the sugar rings in green. Only the nicotinamide portion functions in
redox; the remainder of the molecule, symbolized in the figure as R, selectively binds the
enzyme. The hydroxyl group of R indicated in yellow is either unmodified (NADH, NAD+) or
esterified to a phosphate group (NADPH, NADP+). The extra phosphate provides discrimina-
tion between enzymes that use either NAD+/NADH or NADP+/NADPH, without affecting
the electron transfer itself.
NAD+-assisted hydride extraction from an alcohol is illustrated in Figure 7.2. Electron
flow from the neighboring oxygen atom initiates the movement of the electron pair from
Chapter 7 – Coenzymes 141
TABLE 7.1 Vitamin Precursors of Coenzymes

Vitamin Source Structure Coenzyme
Biotin Eggs, whole grains O
NH Biotin
B7 HN
COOH
Folate Meats, leafy H2N

N N
Dihydrofolate,
B9 vegetables, whole tetrahydrofolate
H
N
N
N
grains OH
H
N COOH
O
COOH
Niacin Cereals, eggs, O NAD+, NADP+

B3 vegetables
NH2
Pantothenate Meats, whole grains OH Coenzyme A

H
B5 HO
N
COOH
H3 C CH3 O
Phylloquinone Leafy vegetables, plant O Phylloquinone

K1 oils
O 3
Pyridoxine Meats, cereals, fruits OH Pyridoxal phosphate

B6 HO OH
H3C N
Riboflavin Milk, eggs CH2OH FAD, FMN

B2
HC OH
HC OH
HC OH
CH2
N N
H3C O
NH
H3C
N
O
Thiamine Whole grains NH2 CH3 Thiamine pyrophosphate

B1 N N +
OH
S
H3C N
the alcohol onto the cofactor. Electron migration through the nicotinamide ring is driven
by the positively charged nitrogen atom, an electron sink. The reaction products are the
carbonyl compound and a proton. Nicotinamide nucleotides, like the adenine nucleotides,
are mobile cofactors. For a steady-state conversion of alcohol to ketone in this example, there
must be separate reactions that carry out re-oxidation of NADH at the same rate as the alco-
hol oxidation.
While we examined only the NAD+ reaction, reactions using NADP+ are chemically indis-
tinguishable. There is an important biological distinction: the binding of distinct enzymes
to the phosphorylated versus nonphosphorylated nucleotides enables two separate redox
poises – NAD+/NADH and NADP+/NADPH – to be maintained in the same cellular water
142 7.3 Redox Coenzymes
TABLE 7.2 Coenzymes

Coenzyme Type Reaction Example Pathway Reactive Portion Descriptive name
REDOX
NADH, NADPH Mobile Lactate dehydrogenase Glycolysis O Hydride donating
H H nicotinamide
NH2
Ubiquinone (Q) Mobile Cytochrome oxidase Electron transport chain O Quinone

Plastoquinone (PQ)
FAD, FMN Bound Glutathione reductase Free radical removal Flavins

N N
O
HN
N
Acyl Transfer
Carnitine Mobile Carnitine acyl transferase Fatty Acid oxidation OH Alcohol
Coenzyme A Mobile Citrate synthase Krebs Cycle CoA-SH Thiol

Lipoate Bound Pyruvate dehydrogenase Glucose oxidation S Disulfide
S
R
CARBOXYLATION
Biotin Bound Pyruvate carboxylase Gluconeogenesis O Cyclic urea
HN NH
Phylloquinone (K1) Mobile γ-glutamyl carboxylase Protein carboxylation OH Benzo-quinol

Menaquinone (K2)
OH
EXCHANGE
Thiamine pyrophosphate Bound Pyruvate dehydrogenase complex Glucose oxidation Ylide
N+
S
Pyridoxal phosphate Bound Transaminase Amino acid oxidation O Carbonyl
N
FIGURE 7.1 Nicotinamide coenzymes. NAD+ and NADP+ (and their reduced counterparts) dif-
fer only in the 2’ hydroxyl group of the adenine portion, which is phosphorylated in NADP+. This
hydroxyl group is indicated in yellow.
FIGURE 7.2 Hydride transfer in the nicotinamide ring.

144 7.3 Redox Coenzymes
space. For example, in the cytosol, these ratios differ by five orders of magnitude; yet their
ratios under standard state conditions are identical.
7.3.2 UBIQUINONE
A second mobile cofactor in biochemistry is ubiquinone.
The structure shown in Table 7.2 is the portion that changes during the reaction. The very
similar plastoquinone is missing only a methyl substitution in the ring and has nine repeat-
ing branched lipid segments instead of ten. The chemistry of ubiquinone and plastoquinone
is identical. Generally, free radicals are more unstable than paired electrons in a molecule.
The ability of ubiquinone to stabilize the free radical is due to the spreading out of electron
density through the aromatic ring.
The sequence of reactions involving ubiquinone is illustrated in Figure 7.3 for the direc-
tion of reduction. The fully oxidized quinone is first reduced by one electron to a radical
anion, in which one oxygen has a single unbound electron. The other oxygen has a full nega-
tive charge. Two protons accompany the addition of the second electron to form the fully
reduced UQH2. The single electron donors (or acceptors when the coenzyme acts as a reduc-
tant) are redox-active metal ions, typically Fe2+/Fe3+, bound in chelates as we will discover in
the chapters on oxidative phosphorylation (Chapter 11) and photosynthesis (Chapter 12). In
these processes, ubiquinone or plastoquinone are mobile cofactors but are confined to the
lipid portion of the mitochondrial or thylakoid membrane.
7.3.3 FLAVIN COENZYMES
Flavins are named for the family Flavius (“yellow-haired”), a series of three Roman emper-
ors in the 1st Century AD. The coenzymes and the proteins they are attached to have a
bright golden yellow color. The flavins are prosthetic groups and stay fixed to their enzymes
throughout the catalytic cycle. As a result, electrons donated to a flavin coenzyme must be
removed to restore its original state. Consequently, a second, mobile cofactor must be associ-
ated with flavin-linked enzymes to remove the electrons.
FIGURE 7.3 Ubiquinone oxidation states

Flavin structures are shown in Figure 7.4. FAD (flavin adenine dinucleotide) has both
adenine and flavin nucleotides; the adenine nucleotide portion, like the one in NAD+ is exclu-
sively for enzyme binding. FMN (flavin mononucleotide) is an abbreviated structure with
only a ribose phosphate attachment. Also shown in Figure 7.4 is the active portion of the
coenzyme.
The flavins are distinctive cofactors because the forward and reverse routes of redox
reactions are asymmetric. As an illustration, consider the reactions shown in Figure 7.5.
FADH2 can be oxidized by donating single electrons, which can be accepted by metal ions.
Figure 7.5A shows the sequential donation of two electrons with an intermediate radical.
Protons are also released sequentially. Figure 7.5B shows the reduction of FAD by direct reac-
tion with NADH. This process is initiated by extracting a proton from an enzyme side chain
(illustrated simply as Enz-BH) and a movement of electron pairs through both the FAD and
NADH rings, forming the products FADH2 and NAD+.
A complete redox cycle involving FAD is presented in Figure 7.6A. In this example, the
electron donor is a redox-active iron protein, represented simply as the iron ion itself. Two
Fe3+ ions are reduced in sequence, converting FADH2 to FAD with the simultaneous release
of protons. The cycle is completed as FAD accepts a hydride from NADH. Overall, the
FIGURE 7.4 Flavin coenzymes
FIGURE 7.5 FADH2 – NAD+ electron exchange. a) FADH2 donates electrons to an exterior
acceptor and is oxidized to FAD b) NADH donates electrons to FAD to regenerate the original
coenzyme form, FADH2
146 7.4 Acyl Transfers
FIGURE 7.6 Alternative representations of FADH2 – NAD+ electron exchange. a) Flavin nucleo-
tides are cycled as net NADH is converted to NAD+ b) The overall redox reaction changes do not
include flavin cofactors as they are regenerated c) The participation of FAD in the overall reaction
is acknowledged in parenthesis.
reaction is shown in Figure 7.6B. Notice that flavins do not appear in this reaction, just as
intermediate enzyme forms do not appear in overall reactions. An abbreviated form of the
reaction, emphasizing the mobile coenzyme role of NADH, is shown as Figure 7.6C. Here,
the emphasis is on the flow of electrons to reduce Fe3+, coupled with the oxidation of NADH.
The participation of the bound flavin cofactor can be indicated by placing it in parenthesis
beneath the reaction arrow. A common representation error is to place flavin cofactors as
apparent mobile cofactors, that is, not showing how they are regenerated but leaving them in
a reduced or oxidized state.
7.4 ACYL TRANSFERS
Coenzymes that participate in acyltransferase reactions engage in ester formation. Most

acyltransferases involve a thioester, often CoA (coenzyme A). There is little new chemistry
involved in the acyltransferase coenzymes. The amino acid side chains of cysteine or serine
share the ability to form esters. As we examine the cofactors in their enzymatic role, it will
become evident why they are required.
7.4.1 CoA
The most common cofactor for acyltransferase reactions is CoA (Figure 7.7). Like the nico-
tinamide and flavin nucleotides, this coenzyme has an adenine nucleotide exclusively used
for enzyme binding, rather than chemical function. Attached to the ADP portion is panto-
thenate, or Vitamin B5 (Table 7.1). An amide link between the pantothenate and mercapto-
ethylamine completes the structure. The sulfhydryl moiety is the only reactive functional
group, with the same chemistry as the alkyl sulfhydryl (–CH2SH) of cysteine.
Fritz Lippman discovered CoA in 1945 as a cofactor needed for enzymatic acetylation
reactions. He assigned the name as an abbreviation for “coenzyme of acetylation.” CoA was
the answer to a long quest for the identity of active acetate, now known to be the CoA ester
acetyl‑CoA, a central intermediate in oxidative metabolism. CoA is also involved in other
acyl formations. Figure 7.8 shows one of these reactions: a long-chain fatty acid is converted
to a fatty acyl-CoA, a step also known colloquially as “activation” of a fatty acid. In the figure,
FIGURE 7.7 CoA. The red portion contains the sulfhydryl, the reactive part. Pantothenate in
blue is the portion not synthesized by humans and is thus required as a vitamin. The adenine
nucleotide is used exclusively for binding, as with flavin and nicotinamide nucleotides.
FIGURE 7.8 Fatty acid activation requires CoA. The conversion of fatty acids into their CoA
esters involves the coenzyme CoA, as well as ATP, AMP, and PPi (pyrophosphate).
FIGURE 7.9 Carnitine acylation. Carnitine participates in the carnitine acyltransferase cata-
lyzed exchange of the thioester of the acyl-CoA for the oxygen ester of acyl-carnitine.
CoA is written in its alternative form, “CoA-SH,” which emphasizes the reactive portion of
the coenzyme structure. Notice that the CoA becomes incorporated into the product and is
only released in a later reaction. This explains its use in place of a cysteinyl side chain of a
protein; the CoA is mobile and can engage in subsequent reactions. Thus, CoA can be con-
sidered an acyl carrier.
7.4.2 CARNITINE
Named for its discovery in meat (Latin: carne, flesh), carnitine is an essential coenzyme for
the oxidation of long-chain fatty acids (Chapter 14). Carnitine forms an ester with the acyl
CoA molecules in the reaction illustrated in Figure 7.9. The enzyme catalyzing this reaction
is known as CAT, for carnitine acyltransferase. The rationale for this transformation is to
provide a molecule that can be transported across the mitochondrial membrane. Unlike the
148 7.5 Carboxylation
coenzymes we have examined so far, carnitine is endogenously synthesized from a lysine side
chain that is subsequently methylated on the γ-nitrogen and hydroxylated and subsequently
released during proteolysis.
Like most of the coenzymes we have studied, carnitine is a mobile cofactor. It is a simple
matter to determine which coenzymes are mobile; they have two forms that appear on both
sides of a net reaction.
7.4.3 LIPOIC ACID
Lipoic acid (Figure 7.10A) is present in an enzyme-bound form (Figure 7.10B) when it par-
ticipates in catalysis. The shorthand form is shown in Figure 7.10C. The disulfide engages in
short-chain acylation reactions, as indicated in Figure 7.11. The conversion of the disulfide
to the acetylated state (step 1) results from a nucleophilic displacement by an acetyl group.
The two-carbon fragment is removed in step 2 by reaction with CoA, the mobile coenzyme
described above. The coenzyme is now in a reduced form. Step 3 is a re-oxidation back to
the disulfide that can re-enter the catalytic cycle by accepting another acetyl group. The re-
oxidation requires to other cofactors: FAD and NAD+ (Chapter 10).
The cyclic nature of bound coenzyme regeneration is evident in Figure 7.11. The purpose
of step 3 is to return the coenzyme to its disulfide form. While lipoamide is not classified as
a redox coenzyme, it does undergo a redox reaction in the course of transferring acyl groups.
Redox involvement is also present with other coenzymes, particularly the folates, as will be
apparent in Chapter 15.
A closely related structure to lipoic acid is asparagusic acid, a compound uniquely syn-
thesized by asparagus. Rather than acting as an intracellular coenzyme, this compound
is secreted extracellularly in this plant to repel its nematode predators (Box 7.1: A Lipoate
Analog in Asparagus).
7.5 CARBOXYLATION
There are three types of biological carboxylation. The first – net incorporation of CO2 into
organic compounds – is catalyzed by the plant enzyme ribulose bisphosphate carbox-
ylase, and does not use a coenzyme (see Chapter 12). A second type requires the vitamin
biotin (Table 7.1), covalently incorporated into its associated enzymes. Biotin-dependent
carboxylation targets small molecules at a carbon adjacent to a carbonyl. The CO2 added to
such molecules is released in a subsequent reaction. The third type uses Vitamin K, a mobile
cofactor for the post-translational carboxylation of proteins at the γ-carbon of glutamate
residues, forming a second carboxyl group that carbon.
FIGURE 7.10 Lipoic acid. a) The free form of lipoic acid before its incorporation into an enzyme
b) The amide form of lipoic acid attached to a lysine side chain of an enzyme c) The abbreviated
active portion of lipoic acid.
FIGURE 7.11 Acylation reactions of lipoic acid. 1) an acetyl group becomes attached to one
of the sulfur atoms to form the thioester. 2) Transfer of the acetyl group to an external acceptor
(usually CoA) leaves the lipoic acid in the sulfhydryl form. 3) The original disulfide form is restored
by reaction with a separate oxidant coenzyme.
Box 7.1 A Lipoate Analog in Asparagus
Nematodes (roundworms) are a common agricultural parasite. Species that infect aspara-
gus are thwarted by a compound that appears to be synthesized uniquely by the shoots
of this plant, known eponymously as asparagusic acid
OH
S
S
Asparagusic Acid
This compound is harmless to humans when ingested; however, it is metabolized to

several volatile sulfur compounds, accounting for the scent of the urine following
ingestion.
7.5.1 BIOTIN
The biotin vitamin (Table 7.1) is incorporated into enzymes that bind it covalently in an
amide bond to the lysine residue of an enzyme. Biotin enzymes have two active sites. In one,
carboxylated biotin is formed from ATP and CO2 (Figure 7.12). In Site 1, bicarbonate – the
hydrate form in equilibrium with CO2 – reacts with ATP to form the acid phosphate, car-
boxyphosphate. A nitrogen atom of biotin displaces the phosphate and forms the adduct
carboxybiotin. In Site 2, a substrate – in this case, pyruvate – reacts with carboxybiotin to
form the product, oxaloacetate, and regenerates the free biotin enzyme form. The enzyme in
this example, pyruvate carboxylase, is further discussed in Chapter 13.
The carbon incorporated by biotin-linked enzymes is released in a subsequent reaction.
The purpose of this carboxylation is to energetically drive the later reaction, rather than to
incorporate net carbon.
150 7.5 Carboxylation
FIGURE 7.12 Biotin dependent carboxylation. An “active CO2” is formed by reaction with ATP
to produce carboxyphosphate. At Site 1 of enzymes using biotin, the cofactor binds the CO2.
The attached biotin-CO2 swings over to a separate region of the enzyme – Site 2 – which is a
distinct active site, where CO2 from biotin attaches to the substrate.
7.5.2 VITAMIN K
The carboxylation reaction assisted by Vitamin K has a single target: the glutamate side chain
of a protein. Protein carboxylation is a type of post-translational modification, distinct from
protein phosphorylation we have considered in the previous chapter because carboxylation
is not reverted by subsequent reactions.
Carboxylation takes place as the protein is being formed and threaded into the ER (endo-
plasmic reticulum) lumen. This modification is thus similar to N-linked glycosylation
(Chapter 5); both alterations in the protein contribute both to driving protein folding and
protein function once it is outside the cell.
The chemistry of the carboxylation reaction is distinct from other forms of CO2 incor-
poration: a proton is removed from a methylene group. This acid–base reaction is unusual
because the removal of a methylene proton requires a very strong base. In water, the stron-
gest base available is the hydroxide ion, which is far too weak to effect the removal of a
methylene proton.
The species required for this task is an alkoxide ion (a carbon-attached oxygen anion),
which has the same pKa as serine (Chapter 5; the hydroxyl group of serine has a pKa of 16).
While we are considering coenzymes abstracted from their protein environment, it is essen-
tial to recognize the importance of the protein. In the present case, a hydrophobic environ-
ment at the active site is critical for the creation of the alkoxide. The generation of the alkoxide
involves the reaction of the benzyl quinone of K1 with oxygen, as outlined in Figure 7.13.
After removing a proton from a phenolic hydroxyl, an oxygen molecule adds across the dou-
ble bond drawn in purple to form the alkoxide (one of the oxygen atoms) and an epoxide
(the other oxygen atom). The alkoxide can extract the hydrogen atom from the C–H bond to
form a carbanion that can subsequently attack CO2 to produce the carboxylated glutamate.
After abstracting the proton, the coenzyme is in its quinone form. Reductants (NADPH)
are needed to restore the original coenzyme (see the chapter addendum for further details).
The carboxylation occurs in the lumen of the endoplasmic reticulum of the cell as the
protein is being synthesized. Several glutamate residues are modified in this manner. The
result of this carboxylation is to form a dicarboxylate that chelates Ca2+. The dicarboxylate
has a relatively low affinity for Ca2+ (binding constants are near 1 mM) consistent with the
FIGURE 7.13 The vitamin K cycle. The quinol form of the top left of the figure first dissociates
a proton, then reacts with O2 to form the reactive alkoxide. An alkyl proton, abstracted from a
methylene group in the substrate, reacts with CO2 to form the carboxylated glutamate residue.
The coenzyme then assumes the quinone form. Two separate reactions are required to restore
the original quinol form to complete the cycle.
approximate Ca2+ concentration within the ER lumen and the extracellular space, the desti-
nation of the modified proteins.
Prominent among the carboxylated proteins are some of those involved in the blood
clotting cascade. Vitamin K is named for this process (German: koagulation); the proteins
involved include proteases that catalyze cleavage and activation of factors that ultimately
lead to the formation of a clot by converting fibrinogen to fibrin. The reason for the carbox-
ylation appears to be two-fold. First, it assists in protein folding as the protein is synthesized
in the ER. Once in the blood, the bound Ca2+ binds low-affinity sites of phosphate (from
phospholipids) on cell surfaces, providing an anchor for the carboxylated protein. Aside from
their involvement in blood clotting, γ-glutamyl-carboxylated proteins are also found in anti-
coagulation proteins (clot resorption), and proteins involved in tissue calcification in the con-
trol of bone formation.
7.6 EXCHANGE COENZYMES
Thiamine pyrophosphate and pyridoxal phosphate participate in enzymatic exchange

reactions. We will see that the mechanisms for transketolase (thiamine pyrophosphate) and
transaminase (pyridoxal phosphate) are both symmetrical, reacting with one substrate and
then repeating the process in reverse to react with a second substrate. Each cofactor also
participates in a wide range of other reactions.
7.6.1 THIAMINE PYROPHOSPHATE
Thiamine pyrophosphate (TPP) is the direct pyrophosphate derivative of the vitamin thia-
mine (Table 7.1). The structure of TPP shown in Table 7.2 highlights its active carbanion.
While carbanions are usually very unstable, this one is stabilized through the resonance
of the thiamine ring. Carbanion formation requires the loss of the proton indicated in
Figure 7.14. An alkyl anion neighboring a cation, such as the positively charged nitrogen
atom in thiamine, is known as a ylid. The charge stabilization of this structure accounts for
the ready proton dissociation.
One reaction of TPP-linked enzymes is the decarboxylation of ketoacids. For example,
yeast pyruvate decarboxylase utilizes the thiazole ring to decarboxylate pyruvate, releasing
acetaldehyde after hydrolysis, regenerating the original form of thiamine. The reaction steps
leading to the release of the CO2 are shown in Figure 7.15. Electron rearrangement of the
thiazole ring is required to release the acetaldehyde portion (see Chapter 9).
152 7.6 Exchange Coenzymes
FIGURE 7.14 Dissociation of thiamine to a stable carbanion. The equilibrium favors the carb-
anion form on the right, with both positive and negative charges in the molecule.
FIGURE 7.15 Decarboxylation of pyruvate assisted by TPP. The ylid form of TPP attaches to
pyruvate, enabling electron movement to decarboxylate the molecule to acetaldehyde.
Thiamine is required in humans for the central step of converting pyruvate into acetyl-
CoA (Chapter 11), and in chemically similar steps in branched-chain amino acid metabo-
lism (Chapter 15). A distinct reaction that utilizes thiamine is the transketolase reaction,
which rearranges sugar molecules in the Calvin Cycle (chapter 12) and the “pentose phos-
phate shunt” (Chapter 13). The overall conversion of two five-carbon sugar phosphates, X5P
(xylulose-5P) and R5P (ribose-5P) into a 3-carbon (GAP, glyceraldehyde-P) and a 7-carbon
(sedoheptulose-7P) product is indicated in Figure 7.16a. The reaction mechanism is outlined
in Figure 7.16b. After an attack on X5P by the ylid, the cleavage between C2 and C3 produces
the first product, GAP. The coenzyme-bound fragment is identical to the intermediate in
pyruvate decarboxylase. However, here the two-carbon fragment is displaced by R5P, regen-
erating the original form of the bound coenzyme and producing the second product, S7P.
7.6.2 PYRIDOXAL PHOSPHATE
Several forms of Vitamin B6, including the structure of Table 7.1, as well as aldehyde and
amine forms, are converted in humans into pyridoxal phosphate
O O
C
OH
2-
O3P
N
N CH3 H
Pyridoxal Phosphate (PALP) PALP, abbreviated structure

FIGURE 7.16 TPP Action in transketolase. The exchange reaction (a) is chemically similar to
pyruvate decarboxylation. In the transketolase reaction (b), after splitting off GAP, the fragment
remaining attached to TPP is then joined to a second substrate, R5P.
This coenzyme is a prosthetic group that participates in exchange reactions like the trans-
ketolase mechanism of thiamine. In the case of pyridoxal phosphate, the enzymes are known
as transaminases.
Transaminase reactions exchange amino and keto groups between substrates and play a
prominent role in nitrogen metabolism (Chapter 15). The essence of the mechanism is the
interaction of a carbonyl and an amine to form an imine (Figure 7.17) known as a Schiff
Base reaction, after its discoverer (Box 7.2: Name Origins: Schiff Base). The amine attacks
the carbon of the aldehyde, forming an intermediate that is successively protonated: water
release produces the imine. The mechanism is another example of a nucleophilic attack with
acid–base catalysis introduced in the previous chapter.
The best-known transaminase is aspartate aminotransferase, known clinically as sGOT
(soluble glutamate-oxalacetate aminotransferase). It is part of most blood sample analyses,
as it is in very high intracellular concentrations, particularly in the liver. Its presence in the
blood indicates damage to liver cells. The overall reaction is:
(7.2) aspartate + a-ketoglutarate oxaloacetate + glutamate
Some of the mechanistic details for the transaminase are presented in Figure 7.18, which
we will name the three iminos, identified by the blue circles of the figure. The first struc-
ture (Figure 7.18a) is the free form of the enzyme, bound in Schiff base to the aldehyde of
PALP. The top row of Figure 7.18 are steps leading to the exchange of this Schiff base for a
different one, engaging PALP with aspartate in a Schiff base (Figure 7.18d). This process is
initiated by nucleophilic attack by the N atom of aspartate (Figure 7.18b). In the next step,
an electron pair from the same N undergoes rearrangement (Figure 7.18c). The formation of
the new Schiff base in Figure 7.18d can be considered a transimidization. The bottom row
of Figure 7.18 shows the use of the electron sink of the pyridine nitrogen of Figure 7.18e in
154 7.7 Metal Ion Cofactors
FIGURE 7.17 Schiff Base formation. The reaction between an amine and a carbonyl group
proceeds by nucleophilic addition, proton addition, and water elimination. The product is an
imine, an important intermediate in several enzymatic mechanisms, although the reaction itself
can proceed nonenzymatically and is freely reversible.
Box 7.1 Name Origins: The Schiff Base
In 1864, Hugo Schiff discovered that aniline compounds (benzylamines) could react with
aldehydes to form imines. The importance of the discovery is indicated by the fact that
it was incorporated into a textbook written by Friedrich Kekulé, himself notable for the
discovery of aromaticity in benzene. Schiff had extensive interactions with the chem-
ists of the 19th century, starting with his doctoral mentor Friedrich Wöhler (urea synthe-
sis, Chapter 1), Stanislao Cannizzaro (alcohol-aldehyde reaction), and Dmitri Mendeleev
(Periodic Table construction). The naming of the imine as a base is more historical than
functional. Schiff designated the imine as a base, although it is not used as a base in
chemical practice nor in enzymatic mechanisms. Still, it was assigned this classification in
the Kekulé textbook, and the name, attached to its founder’s name, remains today.
moving electrons from the α-hydrogen of aspartate, producing the structure of Figure 7.18f.
In the next step, the carbonyl carbon of PALP accepts a proton from the enzyme lysine
group, drawing electrons from the PALP ring nitrogen. This rearrangement produces the
third imine, shown in Figure 7.18g. The water addition (hydrolysis) step leading to the forma-
tion of oxalacetate and pyridoxamine (Figure 7.18h) is the reversal of the Schiff Base reaction
(Figure 7.17). The mechanism shown in Figure 7.18 accounts for only half of the mechanism.
The other half can be visualized by replacing the keto-acid oxaloacetate with α-ketoglutarate,
and reversing all the steps until the amino acid glutamate is released in the reaction from
Figure 7.18c to Figure 7.18a.
Enzymes other than transaminases use PALP. After binding the active aldehyde of the
cofactor, substrates may be decarboxylated (amino acid decarboxylase), converted to a dif-
ferent stereochemical form (D-amino acid racemase), or undergo β-elimination (serine dehy-
dratase). A surprisingly simple use of this cofactor is for the breakdown of glycogen to free
glucosyl units. In this case, the active aldehyde of PALP remains on the enzyme in a perma-
nent Schiff base (glycogen phosphorylase). Here, only the phosphate group participates in
acid–base catalysis (Chapter 14).
7.7 METAL ION COFACTORS
Several ions serve as enzyme cofactors that in some cases resemble coenzymes. Here we
consider the major ions and their roles in enzyme action.
FIGURE 7.18 The Three Iminos mechanism of transamination. Half of the transamination
mechanism focused on the coenzyme PALP is shown in the figure. A simplified overall view is
obtained by focusing on the three imines, or Schiff bases, encircled in blue. The first is in (a) with
PALP attached to an enzyme lysine. A series of electron rearrangements releases the enzyme and
forms a Schiff Base between aspartate (the first substrate) and PALP (d). Further electron move-
ment displaces the Schiff base away from the ring (g) that can be hydrolyzed to release the first
product, the ketone oxaloacetate, and the amino form of PALP. The reactions are then essentially
reversed, except that a different ketone – the second substrate 2-ketoglutarate – reacts with the
amino-PALP.
7.7.1 Mg2+
This ion is abundant both intracellularly as well as extracellularly. It exists biologically only
in the +2 oxidation state and thus is classified as a non-redox active ion. Most cellular ATP is
bound to Mg2+, and it also loosely binds other phosphorylated compounds in the cell. Mg 2+
does exist as a chelate in chlorophyll (Chapter 12).
7.7.2 IRON
Iron is present in bound form in all cases, either in transport proteins that bring the metal
into the cell (e.g., ferritin), in the non-redox active hemes of myoglobin and hemoglobin, or
the redox-active hemes of cytochromes and iron-sulfur proteins (Chapter 11).
7.7.3 COPPER
While more limited in occurrence, redox-active copper ions (+1 and +2 valence) are part of
the mitochondrial respiratory chain (Chapter 11).
7.7.4 Co2+
Cobalt ions appear to be involved in just one type of reaction: the movement of methyl
groups in reactions utilizing the coenzyme cobalamine, or Vitamin B12. Co2+ exists in this
vitamin as a chelate structure resembling heme, known as a corrin ring. A reaction utilizing
this coenzyme is part of lipid metabolism (Chapter 14).
7.7.5 Mn2+AND Zn2+
These ions are commonly found tightly bound to enzymes. They can assist in binding to
substrates and provide an electron sink, like the quaternary nitrogen of NAD+ or pyridoxal
phosphate.
7.7.6 SELENIUM
Humans require trace amounts of selenium, an element in the same group as sulfur. For
some proteins, the amino acid selenocysteine becomes incorporated during synthesis. In
some sense, selenocysteine is a “21st amino acid”, although it occurs in very few protein
chains. Selenium containing proteins are commonly involved in redox processes related to
detoxifying oxygen radicals, such as the enzyme glutathione peroxidase.
7.7.7 DIETARY ESSENTIALS
Our consideration of vitamins and metals as dietary essentials raises the question of exactly
how we know a compound is essential and to what extent. Sometimes the grouping of “vita-
mins and minerals” suggests we can put the essentials together; it is important to keep coen-
zymes separate to understand their distinct biochemical function.
Some vitamin deficiencies lead to known disorders. These include scurvy (lack of Vitamin
C), beriberi (lack of thiamine), and hemorrhage (lack of Vitamin K). For others, it is less clear.
For example, despite the requirement of pantothenic acid for CoA, there are no defined defi-
ciency disorders. The lack of nutritional deficiency disorders for many vitamins is likely due
to their ready availability in the environment.
A related issue is the matter of dosage. For the most part, this is estimated from animal
experiments and extrapolated to humans. Since coenzymes are needed in limited amounts,
either as a pool for the mobile coenzymes, or attached to enzymes for the bound coenzymes,
there is no known reason that large intake would be beneficial. In some cases, large doses
can be toxic.
A final consideration is that a change of state can alter what becomes a dietary essential.
For example, some drugs can interfere with cofactor availability. In the case of a widely pre-
scribed class of drugs known as statins (Chapter 14) taken to lower blood cholesterol levels,
there is a secondary inhibition of the formation of compounds that form branched lipids.
Branched-chain lipids are part of the structure of lipoic acid and UQ so that these coen-
zymes can become dietary requirements in the presence of statins.
REVIEW QUESTIONS
1. The single-electron reduced flavin nucleus can be drawn in distinct ways and is often
written in different forms than in the figures shown. Why is that possible?
2. Why isn’t Fe2+ or Fe3+ considered a coenzyme?
3. The same molecule of ATP can interact with hundreds of enzymes in a cell. How many
enzymes can a given molecule of FAD react with?
4. Which cofactors transfer hydride ions?
5. Why don’t NADH and NADPH generally bind the same enzymes?
6. Why is it difficult to discover a vitamin deficiency in many cases?
7. Most acyl transfer reactions involve thioesters rather than (oxygen) esters. Why is that
the case?
8. The alkoxide formed in the action of Vitamin K is a stronger base than OH- (hydroxide
ion). Since no base can be stronger than the hydroxide ion in water, how can this be
achieved?
9. An enzyme, using only its side chain amino acids, could not perform the following
transformations. Identify the coenzyme(s) that allows each of them:
a) Abstraction of an alkyl hydrogen
b) Exchange of amines and keto acids
c) Carbanion formation for nucleophilic attack
d) Hydride transfer
e) ATP-dependent carboxylation
f) ATP-independent carboxylation
g) Exchange of acyl groups
h) Reduction of a carbonyl group
i) Reduction of a metal ion
10. Which metal cofactors are redox-active and found in chelates in proteins?
11. Why isn’t selenocysteine always considered a 21st amino acid?
CHAPTER 7 ADDENDUM: THE VITAMIN K CYCLE
Phylloquinone was described in the chapter text as a mobile coenzyme, but it is unusual in
regenerating its original form. Here we take a more comprehensive look at the pathway for
regeneration of the active form of this coenzyme.
Our task is to fill in the omitted steps of Figure 7.13. We abbreviate the O2-dependent
carboxylation reaction described in Figure 7.13 as
O
OH
(CA7.1) OH O
Hydroquinone + Prot-Glu + O2 + CO2 → Epoxide + Prot-Gla
where Prot-Glu represents a glutamate residue of the protein, and Prot-Gla is the standard
abbreviation for a γ-glutamyl-carboxylated protein residue. The carboxylation reaction
appears as the top line of Figure A7.1, part of a cycle in which the coenzyme is regenerated.
Comparing the epoxide to the hydroquinone, it is evident that the regeneration must consist
of reduction steps. These are shown leading to the formation first of the quinone structure,
which is also the vitamin form, and then the hydroquinone. Not indicated is the donor of the
reducing equivalents, which surprisingly remains uncertain. The enzymes are all integral
membrane proteins and measured in vitro using artificial acceptors. It is established that
sulfhydryl groups are involved in the reductions in both cases, so that it is likely that flavin
coenzymes are the proximal donors.
Also indicated in Figure A7.1 are the sites of inhibition exerted by warfarin, which was
designed as an analog of coumarin
O O
Coumarin
a molecule discovered in spoiled clover that caused bleeding when consumed by cattle.
Currently, warfarin is a widely used drug in blocking blood clot formation as it reduces the
158 Key Terms
FIGURE A7.1 Ancillary enzymes for Vitamin K Regeneration. Two separate reactions are
required to regenerate the hydroquinone form of Vitamin K. Each requires reductants that have
yet to be identified. The first step is epoxide reductase; the second is Vitamin K reductase. Both
enzymes are inhibited by warfarin, which explains why the drug inhibits protein carboxylation
and thus the activity of three enzymes of the blood clotting cascade.
ability for gla residues required in some of the proteases involved in the clotting cascade.
Warfarin was developed at the University of Wisconsin; the name is derived from Wisconsin
Research Alumni Foundation.
KEY TERMS
biotin
CoA
cobalamine
coenzyme
coproducts
cosubstrates
electron sink
mobile cofactors
phylloquinone
prosthetic group
pyridoxal phosphate
pyruvate carboxylase
radical anion
Schiff Base
statins
transaminases
transketolase
Vitamin K
warfarin
ylid
BIBLIOGRAPHY
K.L. Berkner. The Vitamin K-Dependent Carboxylase. Annu. Rev. Nutr. 25 (2005) 127–149.
D.J. Chitwood. Phytochemical Based Strategies for Nemotode Control. Annu. Rev. Phytopathol. 40
(2002) 221–249.
M. Halder, P. Petsophonsakul, A.C. Akbulut, A. Pavlic, F. Bohan, E. Anderson, K. Maresz, R. Kramann,
L. Schurgers. Vitamin K: Double Bonds Beyond Coagulation Insights into Differences between
Vitamin K1 and K2 in Health and Disease. Int. J. Mol. Sci. 20 (2019).
M.O. Hottiger, P.O. Hassa, B. Lüscher, H. Schüler, F. Koch-Nolte. Toward a Unified Nomenclature for
Mammalian Adp-Ribosyltransferases. Trends Biochem. Sci. 35 (2010) 208–219.
N. Kresge, R.D. Simoni, R.L. Hill. Fritz Lipmann and the Discovery of Coenzyme A. J. Biol. Chem. 280
(2005) e18.
K. Lambert, S. Bekal. Intoduction to Plant-Parasitic Nemtatodes. https://www.apsnet.org/edcenter/
disandpath/nematode/intro/Pages/IntroNematodes.aspx (2002).
SC. Mitchell, R.H. Waring. Asparagusic Acid. Phytochemistry 97 (2014) 5–10.
NS. Punekar. Enzymatic Oxidation–Reduction Reactions. Enzymes: Catalysis, Kinetics and
Mechanisms. Springer, Singapore (2018) 385–402.
M.A. Rishavy, K.L. Berkner. Vitamin K Oxygenation, Glutamate Carboxylation, and Processivity:
Defining the Three Critical Facets of Catalysis by the Vitamin K-Dependent Carboxylase. Adv.
Nutr. 3 (2012) 135–148.
M.J. Shearer, T. Okano. Key Pathways and Regulators of Vitamin K Function and Intermediary
Metabolism. Annu. Rev. Nutr. 38 (2018) 127–151.
JK Tie, D.W. Stafford. Functional Study of the Vitamin K Cycle Enzymes in Live Cells. Elsevier,
Amsterdam (2017) 349–394.
USDA. USDA Vitamin Information. U.S. Dept. of Agriculture web site.https://www.apsnet.org/ed
center/disandpath/nematode/intro/Pages/IntroNematodes.aspx (2020).
Metabolism and Energy 8
Metabolism is closely connected to energy changes. We intuitively understand that food
breakdown in the body produces the energy that drives movement and maintains tissues. A
deeper understanding requires a study of thermodynamics, the science of energy changes.
8.1 ORIGINS OF THERMODYNAMICS
Thermodynamics was developed in the 19th century to answer the pressing question of the
era: how to build a better steam engine. This machine was the driving force of the Industrial
Revolution, which changed the world from an agrarian to a city-based economy.
Because of these historical origins, thermodynamics equations use terms more akin
to steam engines (e.g., heat and work) than to biochemical reactions. Still, the variables
in steam power – pressure, volume, and temperature – are concrete and easily grasped.
Additional variables are needed in chemistry, such as changes in chemical concentration
(chemical potential) and changes in electrical potential (voltage).
The principles of thermodynamics – or laws – have two origins. The first is phenomeno-
logical, drawn from experience with no proof other than plausible reasoning that has yet to
be found to have any internal contradictions. The second origin is statistical, deriving equa-
tions from the averaged behavior of large collections of molecules. Happily, the two views
arrive at identical conclusions, providing confidence in the concepts of thermodynamics.
8.2 FIRST LAW OF THERMODYNAMICS
8.2.1 HEAT AND WORK
The first law of thermodynamics is a conservation principle concerning the internal energy
of a system. A system (Figure 8.1) is simply a portion of the universe (i.e., all that exists)
that we wish to study. A boundary line divides the system from its surroundings (i.e., every-
thing in the universe apart from the system under study). Both material and nonmaterial
exchanges can occur across this boundary between the system and the surroundings.
The nonmaterial exchanges across the boundary of Figure 8.1 are themselves representa-
tions of energy. Experience has shown that they can be divided into two distinct forms: heat
and work. These exchanges, along with a sign convention, are illustrated in Figure 8.2:
Heat (q) transferred from the surroundings to the system is positive.

Work (w) produced by the system is positive.
Heat and work are different forms of energy; they may be interconverted, and both are mea-
sured in joules (Box 8.1).
It is common to confuse heat with temperature. Temperature is a measure of how hot
an object is relative to another; it results from the average kinetic energy of its molecules.
Heat is an energy exchange (or flow) between objects of different temperatures, always in the
direction of high temperature to low temperature. The system in Figure 8.2 cannot contain
161
162 8.2 First Law of Thermodynamics
FIGURE 8.1 Thermodynamic definitions. A Venn diagram of the technical meanings of some
fundamental terms. The system is the object of our study. Everything that influences it is called
the surroundings. The system and surroundings are separated by a boundary that defines the
outer limits of the system. Practically speaking, only the immediate surroundings are significant.
The universe consists of both the system and the surroundings.
FIGURE 8.2 Sign conventions of heat and work. Heat transferred from the surroundings to the
system is positive; work transferred from the system to the surroundings is positive.
heat; however, after the flow of heat, there will be a change in system energy, which is called
the internal energy.
The other factor that affects the internal energy is work. Work can be visualized for mol-
ecules as translational, vibrational, or rotational energy. After work has been exerted on or
by a system, there is a change in its internal energy.
It is one thing to state that heat and work are nonmaterial flows across the system bound-
ary. It is another to comprehend what sort of entities these are. We might describe them as
“birds of passage”, meaning that we understand their effects rather than their intrinsic quali-
ties. Not only is a system unable to contain heat, but it also unable to contain work.
We are now in a position to state the first law of thermodynamics:

(8.1) DE = q - w
The term ΔE symbolizes the change (indicated by the delta symbol) in internal energy (E) as
a system moves from one state to another. The notion of a state itself is a technical term in
Chapter 8 – Metabolism and Energy 163
Box 8.1 Joules, Calories, and Food Calories
The units of energy for chemical reactions are joules. One joule (J) is the amount of work
produced by a force of 1 newton over 1 meter (which is about the force of an apple
dropped to the ground); the units are kg-m2/s2. These KGS units can be easily converted
to electrical ones, where 1 J is the work of moving 1 coulomb of charge through a poten-
tial difference of 1 volt. The well-known calorie (cal) unit is based on the heat aspect of
energy: the amount of energy needed to raise 1 g of water 1°C (strictly speaking, the
temperature is raised from 14.5°C to 15.5°C). There are 4.18 J in 1 cal.
Food calories are equivalent to 1000 cal or 1 kilocalorie (kcal). However, they are not
precisely comparable because nutrition databases make corrections for foodstuffs that
are not entirely digestible. Because calories are so closely related to nutrition, this unit has
yet to be fully displaced by the joule.
Box 8.2 Why Spanish Speakers Understand Thermodynamic States
Words used in scientific context are often subtly different from those used in everyday
speech. Languages often have multiple meanings for the same word, or different words
may have the same meaning. The word state would seem straightforward, but it must be
clearly differentiated from variables that are nonstate ones, such as heat and work. One
way to understand the underlying meaning of state is to turn to the Spanish language
words for the English “to be”. Unlike English, Spanish has two separate verbs: ser and estar.
Both are translated as “to be”, but no Spanish speaker would confuse the two or use them
interchangeably. Ser means a permanent identity, such as soy biochemist (I am a biochem-
ist). Estar means a situation that can change, for example, estoy in el laboratorio (I am in
the lab). Later, you might say estoy in el salon (I am in the lounge). This use of two verbs
separates identity from situation; it is the latter that is the essence of state. More than that,
it is the origin of the term itself: the noun form estados means states. The change of state
from one condition to the next is the prime concern of thermodynamics. The details of
how that change is brought about is an issue of kinetics, not thermodynamics. This lan-
guage detour underscores that in the process of making a change of state, the system
identity is unchanged.
thermodynamics (see Box 8.2), meaning a situation that is characterized by a small number
of state variables, such as temperature (T), pressure (P), volume (V), and the number of
moles (n). A system can be said to be in a specific state once all of those variables are defined;
let us call that state-1. If we change some of these variables, we have a new state, called state-
2. Moving from state-1 to state-2 might be done in different ways, but the final result is the
same. The change in state for functions like ΔE is therefore path-independent.
Unlike ΔE, both q and w are path-dependent. However, the remarkable statement of
the first law is that the combination of these variables determines the internal free energy
ΔE, which is itself path-independent. This quality is essential for us to analyze changes in
energy for reactions and pathways. A slightly different quantity than internal energy, called
enthalpy, is important as it is more easily measured in the laboratory.
8.2.2 ENTHALPY
Suppose that our system can only perform expansion work (called PV work where P is pres-
sure and V is volume). The work is:

(8.2) w = D ( PV ) = PDV + VDP
164 8.3 Entropy and the Second Law of Thermodynamics
where the sign is positive as the system is doing work. If we consider only the situation where
pressure is constant (i.e., ΔP = 0), then

(8.3) w = PDV
Under constant pressure conditions, the heat is designated q p and is path-independent. The
overall equation for the first law then becomes:

(8.4) DE = q P - PDV
Rearranging,

(8.5) q P = DE + PDV
We define qP as the enthalpy, an invented word meaning “heat content”. Usually, we use the
term ΔH in place of qP:

(8.6) DH = DE + PDV
It is the enthalpy change that is used to predict whether a system gives off heat (exother-
mic, ΔH < 0) or takes up heat (endothermic, ΔH > 0) from its surroundings. Actually, in
biochemical systems, ΔV is also near zero. Thus, there is little practical difference for our
purposes between internal energy and enthalpy. The data that we use are entirely derived
from enthalpy values, so ΔH is used here.
Enthalpy changes in chemical reactions involve alterations in translation, vibration, and
rotation, all of which contribute to bond energy. Values of ΔH for different processes can be
simply added together. In fact, additivity is a property of all state functions. Thus, we can add
up enthalpy changes for reactions, or sets of reactions, and arrive at enthalpy values for reac-
tions that cannot be determined individually.
Often, an exothermic reaction (ΔH < 0) is one that is favored; that is, it reacts from left to
right as the chemical equation is conventionally written. We commonly experience dramatic
examples of this type of reaction such as in explosions, leaving an expectation that exo-
thermic reactions might predict the direction for reactions. Consider three simple reactions
involving dissolving salts in water, all of which take place in the direction written, along with
their associated enthalpy changes:
H2SO4 + H2O → dissolved salts ΔH < 0

NaCl + H2O → dissolved salts ΔH = 0
KCI + H2O → dissolved salts ΔH > 0
All three of these events take place in the direction written, and yet only the dissolution
of sulfuric acid is exothermic. We must conclude that enthalpy cannot reliably predict the
direction of reactions.
8.3 ENTROPY AND THE SECOND LAW OF THERMODYNAMICS
Entropy (S) is the missing factor needed to predict the direction of chemical reactions. The
second law of thermodynamics states that the entropy change in the universe for any process
is greater than zero:

(8.7) DSuniv > 0
The popular notion that entropy is a synonym for “randomness” does not quite capture the
meaning of the term and is also difficult to translate into a chemical driving force. In order
to clarify the notion, we will explore two different views, one from classical thermodynamics
and the other from statistical thermodynamics.
8.3.1 ENTROPY AS A RATIO OF HEAT TO TEMPERATURE
The entropy concept arose from a study by Sadi Carnot in 1824 to determine the theoretical
maximum efficiency of the steam engine. While Carnot himself did not identify entropy, we
can now assign this function to the ratio of reversible heat produced to the temperature of an
engine that is 100% efficient (the Carnot engine):

(8.8) DS = q rev /T
where qrev is the reversible heat change (the maximum that can be transferred between bod-
ies), and T is the temperature. Like qP, qrev is a state variable. For any real processes – that is,
one in which an engine performs work – it was found that:

(8.9) q/T > 0
This is equivalent to Equation (8.7), the second law of thermodynamics. The fact that entropy
is a ratio of heat to temperature means that, when the temperature is high, a flow of heat into
a system will produce only a small increase in entropy. At lower values of temperature, a heat
change will produce a greater entropy change. However, the statistical approach to entropy
provides a clearer insight into the nature of entropy.
8.3.2 ENTROPY AS A STATISTICAL DISTRIBUTION OF STATES
Ludwig Boltzmann showed that the distribution of molecular energies resembles the Bell
curve. Using statistical insights, he developed an equation for entropy:

(8.10) DS = k × ln W
where S is the entropy; k is a “molecular gas constant” (the molar gas constant (R) divided
by Avogadro’s number (NA)), and W is the number of ways a system can be arranged.
Boltzmann’s insight was to recognize that entropy is a statistical phenomenon. An increase
in the number of possible states increases the entropy. The greater dispersion a reaction
produces in the universe, the greater the entropy. In popular belief, randomness or messi-
ness is equated with entropy. The number of arrangements that appear to be random or
messy is greater than those that seem regular or neat; that is the essence of the concept of
entropy.
This insight allows us to appreciate the role that entropy plays in chemical reactions.
Consider the general case of a reaction that splits the molecule AB into the species A and B:

(8.11) A - B ® A + B
A and B are joined together in the substrate, but separate in the products. Clearly, the prod-
ucts have more degrees of freedom. This increased spatial distribution is a favorable (positive)
change in entropy for the reaction. However, the second law only predicts reaction direction
for a change in the entropy of the universe, not the system. We need a function that can
predict reaction direction that is composed entirely of system variables. That function is the
free energy.
8.4 FREE ENERGY
The free energy is a new state function that combines the first and second laws of thermo-
dynamics. The defining equation is:

(8.12) DG = DH - TDS
166 8.4 Free Energy
Unlike the second law, which is a statement about the universe, the free energy equa-
tion applies to the system under consideration, making it useful for reactions (see chapter
Addendum for a derivation). A reaction is considered favorable (conventionally written in the
left-to-right direction) when:

(8.13) DG < O
A reaction for which ΔG < 0 is called exergonic (avoiding the term spontaneous; see
Box 8.3). The contributions to ΔG are:
ΔH: A heat term; the energy of bond formation or bond breakage

ΔS: An entropy term; the probability or arrangement factor
If a reaction is exergonic and the heat term is dominant (i.e., ΔH is very large and negative),
then ΔH will be the major factor in determining ΔG, and we say that the reaction is enthalpy-
driven. The binding, therefore, is very favorable, and it is quantitatively more important than
the entropy. A dominant ΔH term is a common situation in exothermic reactions. If a reac-
tion is exergonic and the ΔS term is numerically dominant (i.e., ΔS is very large and positive,
such as when KCl dissolves in water or lipid bilayer formation), then the reaction is said to
be entropy-driven.
Not all reactions are exergonic. There are two other possibilities for the value of ΔG. First,
if the reaction is not possible under our defined conditions, then:

(8.14) DG > O
and the reaction is endergonic. An endergonic reaction is not possible thermodynamically,

but the reverse reaction is exergonic because it has the same numerical value for ΔG as the
forward reaction but the opposite sign.
Box 8.3 Word Origins: Spontaneous
In our discussion of the reaction direction, the phrasing would have been more concise if
the word spontaneous were used to indicate the direction that a reaction takes in nature.
In fact, this word has wide popularity and is found in virtually all thermodynamic treat-
ments, from textbooks to specialty monographs. However, it is almost conspicuously
absent in the present treatment because it is a word that tends to confuse rather than
clarify our understanding of thermodynamics.
The most common literature meaning for spontaneous is “of unknown origin”. This is
the definition behind the phrase spontaneous generation, a discarded idea of 19th-cen-
tury biology that organisms can emerge fully formed with no known precursor. Hence,
if you leave fresh meat out in the open air, maggots will appear. This meaning is closer
to “out of nowhere”, another sense of the word spontaneous. There is also a strong
time sense, in that spontaneous events appear to happen very quickly rather than over
long stretches of time. It is also used to introduce mystery because the spontaneous is
unknowable. When the word is applied to reactions, the meanings are equally rich. In
one case, it merely means that a reaction will occur – that is, spontaneous is synonymous
with exergonic (ΔG < O). Yet another common use of the word is as a synonym for non-
enzymatic: reactions occurring without the assistance of an enzyme are “spontaneous”.
It is hard to dislodge from one’s mind the sense that spontaneous reactions must be
faster than non-spontaneous ones. This meaning of the word is a confusion of kinetics
(a time-based concept) with thermodynamics (an energy-based concept) in the study of
reactions. Most educators believe using the word spontaneous provides insight beyond
simply saying exergonic. Because of its rich literary meanings, it detracts from under-
standing reaction direction. The widespread use of the word spontaneous is one reason
many students find thermodynamics difficult.
The other possible value for the free energy change is:

(8.15) DG = 0
In this case, neither the forward nor the reverse reaction is favored. Equation (8.15) is another
definition of equilibrium and demonstrates that equilibrium status and free energy changes
are intimately related.
8.5 STANDARD FREE ENERGY
Standard conditions are conventions that permit laboratories to report consistent results.
All concentrations are set to 1 M, and pressures to 1 atm. While not part of the definition,
it is usually assumed that the temperature is 25ºC or 298K; temperature values are always
included in tables of thermodynamic data. A modification to standard states used by bio-
chemists is that the pH is set to 7; if the hydrogen ion concentration were 1 M, the pH would
be zero (see Box 8.4: Standard Thermodynamic Values).
Changes in enthalpy, entropy, and free energy at standard state are indicated by append-
ing a superscript degree symbol: ΔHº, ΔSº, and ΔGº, respectively. The standard free energy
change (ΔGº) is of particular interest and can be related to the equilibrium constant (Keq).
To show this relationship, we begin with the following equation:
(8.16) DG = DGo + RT ln
[C ][D]
[ A ][B]
which relates the free energy change (ΔG) to the standard free energy change (ΔGº) and
the concentrations of the reactants ([A] and [B]) and products ([C] and [D]) for the general
reaction:

(8.17) A + B C + D
Box 8.4 Standard Thermodynamic Values
The choice of conditions for a standard state reflects two distinct needs. The first is to
approximate laboratory conditions. The pressure is 1 atm at sea level, and room tempera-
ture is close to 25°C (the usual temperature, although not itself part of the definition of
standard conditions). The second need is to simplify the relationship between standard
free energy and the equilibrium constant; hence, all concentrations are 1 M. The argu-
ment of the logarithm term becomes 1, and the logarithm itself is zero.
In what is called “biochemical standard state”, however, hydrogen ion concentration
is taken as 10 −7 M, so pH = 7 (which is much closer to that found in biological systems).
Typically, references include a prime after the superscript zero to indicate this change.
While the values used in this book also are taken from the sources that fix pH at 7, this
stylistic change was not implemented because it complicates the text and focuses the
student on a trivial alteration rather than the main point of standard free energy. There
is another complication with making this change: it invalidates the standard free energy
relationship to Keq. It can be argued that this is just a correction, and we can take this
even further and have all of the reactant concentrations set to much lower values, such
as 0.1 mM, which is typically much closer to physiological values. However, this would still
not produce correct numbers. Instead, it avoids the critical point that there is a distinction
between standard states, equilibrium states, and actual states that must be grasped. This
distinction is more important than setting states closer to actual ones.
Finally, there is a separate standard that is used for the analysis of gasses: the STP,
which means Standard Temperature and Pressure. This standardization fixes only the
temperature at 298K and the pressure at 1 atm. Under these conditions, most ordinary
gases are near-ideal and obey the ideal gas law, PV = nRT.
168 8.6 Nonstandard Free Energy Changes
Recall from Section 8.4 that equilibrium exists when ΔG = 0. Applying this condition to
Equation (8.16) gives Equation (8.18):
0 = DGo + RT ln
[C ][D]
(8.18)
[ A ][B]
Rearranging slightly and adding the “eq” designation to the concentrations to emphasize that
they are equilibrium concentrations:
[C ]eq [D]eq
(8.19) DGo = -RT ln
[ A ]eq [B]eq
The explicit equilibrium notation in Equation (8.19) leads to an alternative definition for ΔGº
because the terms inside the logarithm argument constitute the equilibrium constant. We
can rewrite Equation (8.19) as:
(8.20) DGo = -RT ln K eq
The direct relationship between the standard free energy change and the equilibrium con-
stant exposes the relationship between substrate and product concentrations and the free
energy for a reaction.
8.6 NONSTANDARD FREE ENERGY CHANGES
Under cellular conditions, reactions are at steady-state rather than at equilibrium.

Nonequilibrium reactions are described by Equation (8.16):
(8.16) DG = DGo + RT ln
[C ][D]
[ A ][B]
If we replace ΔGº with −RT∙lnKeq we can rewrite Equation (8.16) as:
(8.21) DG = -RT × ln K eq + RT × ln
[C ][D]
[ A ][B]
The concentration terms in this equation can be defined as:
Q=
[C ][D]
(8.22)
[ A ][B]
where Q is the mass action ratio. Q is the ratio of the product to the substrate for a reaction
that corresponds to a specific physiological steady-state condition. Making this substitution
into Equation (8.21), we arrive at:

(8.23) DG = -RT × ln K eq + RT × ln Q
which is an expression of the nonstandard (actual) free energy change for a reaction in terms
of the equilibrium constant (Keq) and the mass action ratio (Q). For a reaction in cells to
proceed in the forward direction, it is necessary that ΔG < 0. From inspection of Equation
(8.23), the requirement is satisfied if:
(8.24) Q < K eq
We will explore this point further in Section 8.7.

TABLE 8.1 Standard and Actual Free Energies

Situation ΔG ΔGº
Equilibrium 0 −RTlnKeq
Standard Conditions ΔGºrxn ΔGºrxn
Actual Cellular Conditions ΔGrxn −RTlnKeq
If a reaction is endergonic, then ΔG > 0. It follows that in this case, Q > Keq. Such a reac-
tion will not proceed, except in the reverse direction. ΔG = 0 for a reaction at equilibrium,
which is equivalent to Keq = Q.
It is worth pausing at this point to carefully consider the relationship between the equi-
librium, the standard state free energy change, and the actual free energy change (Table 8.1).
While ΔG = 0 at equilibrium, the value of ΔGº is decidedly not zero, although its value can
be calculated from the Keq. Note that neither standard conditions nor equilibrium conditions
provide any information about the free energy under cellular conditions.
8.7 NEAR-EQUILIBRIUM AND METABOLICALLY

IRREVERSIBLE REACTIONS
Reactions that take place in cells are part of sequences called pathways. All reactions in a
pathway proceed at the same rate, and all reactions have ΔG < 0 (i.e., all are exergonic).
From an energetic standpoint, it is useful to divide individual metabolic reactions tak-
ing place in cells into two groups. One of these – by far the largest class – is only slightly
displaced from equilibrium. We can compare Q and Keq by using Equation (8.23). For any
cellular reaction in a pathway, it must be the case that:
(8.24) Q < K eq
but when Q is close to Keq, usually within an order of magnitude or two, we say that the reac-
tion is near-equilibrium. Because most reactions fit into this category, we can consider this
the typical case for cellular reactions, so we expect to find that Q approximates Keq, the equi-
librium ratio. There are three consequences of this observation. First, the reaction is sensitive
to changes in substrate or product concentrations. Secondly, it is possible to run reactions
of this class in reverse because the concentrations are close to their equilibrium values. If
the reaction is running in the reverse direction, it becomes part of a separate pathway. A
third conclusion we can reach concerning near-equilibrium enzymes is that these steps are
unlikely sites of external control, that is, of regulatory behavior. In Chapter 6, we found that
enzymes are analyzed by measuring v, the initial velocity. Since product formation is set
to zero in initial velocity analysis, the reaction must be unidirectional. Therefore, while a
near-equilibrium enzyme may be sensitive to modulators, such as inhibitors, during in vitro
kinetic analysis, we cannot extrapolate this to the physiological situation. Near-equilibrium
enzymes are present at high concentration – which is how they can catalyze the reaction
close to the Keq. Thus, it would require correspondingly high concentrations of an inhibitor
to effect regulation in vivo.
The second class of enzymes has Q values much less than Keq. These reactions are met-
abolically irreversible because they are substantially displaced from their equilibrium
positions. As a result, these reactions cannot run in the reverse direction under cellular
conditions. Metabolically irreversible reactions are invariably the sites of metabolic control
through allosteric or covalent modification. The cellular content (i.e., protein concentration)
of enzymes that catalyze metabolically irreversible reactions is relatively low. Metabolically
irreversible enzymes form bottlenecks, or rate-limiting points, of metabolic pathways.
Determining whether a reaction is near-equilibrium or metabolically irreversible requires
a comparison of Q with Keq. The best-characterized enzymes are in the glycolytic pathway
(Chapter 9), for which extensive data sets are available for many cell types.
170 8.8 ATP
8.8 ATP
Adenosine 5’-triphosphate (ATP) is the central energy intermediate of metabolism. In order

to analyze it from a thermodynamic view, we will start by considering standard free energy
values (Box 8.4) for ATP hydrolysis.
Figure 8.3 shows the hydrolysis reactions of ATP, summarized in Table 8.2, along with
the corresponding ΔGº values. There is a smaller change in ΔGº for the hydrolysis of AMP to
adenosine than for the hydrolysis of ATP to adenosine diphosphate (ADP) or the hydrolysis
of ADP to adenosine monophosphate (AMP). In all of these reactions, there is an increase in
entropy because the substrate molecule is split into two product molecules. This increased
translational freedom accounts for the favorable entropy contribution. Two other effects
explain the lower standard free energy change of AMP hydrolysis. First, when neighboring
phosphoryl groups are separated, the repulsive interactions from the negative charges on
their oxygen groups are relieved, a process that is not present in AMP hydrolysis. Second,
the phosphate groups have more resonance possibilities in the products. This feature is not
present for AMP hydrolysis because one of the products is adenosine, which has no increased
resonance in the product.
In cells, the electrical repulsion of neighboring oxygens is greatly lessened by chelation
with Mg2+ (Figure 8.4). Mg2+ binds so strongly that all reactions involving ATP actually use
MgATP as the reactant (except for one: the mitochondrial ATP translocase (Chapter 11)).
Calculating standard and actual free energy changes for ATP reactions is complicated, in
part because Mg2+ chelation alters the values.
FIGURE 8.3 Hydrolysis reactions of adenine nucleotides
TABLE 8.2 Standard Free Energies

of Adenine Nucleotide Hydrolysis
Reaction ΔGº, kJ/mol
ATP + H2O ⇌ ADP + Pi −30
ADP + H2O ⇌ AMP + Pi −32
AMP + H2O ⇌ Adenosine + Pi −13
FIGURE 8.4 MgATP
FIGURE 8.5 Phosphoryl vs. phosphate
Two other factors must be considered before we can transfer these values to cellular con-
ditions. First, measuring actual concentrations of ADP and AMP generally requires indirect
analysis in living cells because most of the ADP and AMP are bound to proteins. It is only
the free nucleotide concentration that is important thermodynamically, so measuring total
values is misleading. Studies have been conducted to correct for these difficulties, and it has
been found that the cellular ΔG is about −60 kJ/mol in the cytosol of muscle and liver cells if
the reactants are arranged to form the ratio [ATP]/[ADP][Pi]. The second factor to consider
is that ATP hydrolysis does not occur physiologically. In fact, it is the phosphoryl rather than
a phosphate that is transferred (Figure 8.5). Nonetheless, it is useful to compare hydrolysis
reactions and their corresponding ΔGº values to impart ideas of inherent energy in ATP and
to understand energy coupling, our next topic.
8.9 ENERGY COUPLING WITH ATP
Energy coupling is the joining of a process that is endergonic in isolation with one that is
exergonic to produce a combined process that is exergonic overall. We will explore this con-
cept using the asparagine synthetase reaction:

(8.25) Aspartate + ATP + NH 3 Asparagine + ADP + Pi
The reactions and their standard free energy values are shown in Table 8.3. The first entry
(a) is the hydrolysis of asparagine to produce aspartate and NH 3, which has a negative
ΔG º. However, the overall reaction has aspartate as substrate and asparagine as product,
so we are interested in the reverse reaction, which is entry (b) in Table 8.3. The next line
of the table takes the ATP hydrolysis equation from Table 8.2, which has a very negative
TABLE 8.3 Energy Coupling in Asparagine Synthetase

Reaction ΔGº, kJ/mol
(a) Asparagine + H2O ⇌ Aspartate + NH3 −14
(b) Aspartate + NH3 ⇌ Asparagine +14
(c) ATP + H2O ⇌ ADP + Pi −30
(b)+(c) Aspartate + NH3 + ATP ⇌ Asparagine + ADP + Pi −16
172 8.9 Energy Coupling with ATP
FIGURE 8.6 Asparagine synthetase mechanism. The intermediate steps above the line show
the aspartyl phosphate intermediate. Below the line, electron flows are indicated by the curved
arrows.
standard ΔG º. Since standard state functions are additive, we can form the desired reaction
of Equation (8.25) by adding (b) and (c), shown in the last line of Table 8.3. This calculation
shows that the overall reaction is exergonic even though direct asparagine formation (b)
is endergonic.
While the use of standard free energies provides a sense of the energy coupling, we must
be careful not to conclude that ATP hydrolysis in cells can drive asparagine formation. As
noted above, there is no ATP hydrolysis in cells. The actual mechanism for the asparagine
synthetase reaction is shown in Figure 8.6. Note that it involves two nucleophilic substitu-
tions: first, an acid phosphate is formed from at the γ-carboxyl group of aspartate. Next, the
acid phosphate is displaced by ammonia. The enzymatic pathway provides a mechanism for
how ATP bond energy is utilized to activate an otherwise stable carboxylate group to form
the amide product.
The fact that we can deconstruct a reaction into one part positive ΔGº and one part
negative ΔGº, and that it has a negative ΔGº overall is sometimes called energy coupling.
In asparagine synthetase, ATP hydrolysis appears as part of the thermodynamic calcula-
tion and appears to drive an unfavorable process of ammonia incorporation. In the enzy-
matic mechanism (Figure 8.6), there is no ATP hydrolysis. Instead, a phosphoryl group from
ATP forms an acid phosphate with the carboxyl group of the substrate. NH3 subsequently
replaces the phosphoryl group. Thus, there are two distinct meanings of energy coupling:
a thermodynamic one under standard conditions, and a kinetic one that applies to cellular
conditions.
ATP can also be used in energy maintenance rather than coupling. Three examples are
creatine phosphokinase, nucleotide kinase, and adenylate kinase reactions.
8.9.1 CREATINE PHOSPHOKINASE
Creatine phosphokinase catalyzes the reaction of creatine with ATP to form creatine phos-
phate and ADP (Figure 8.7). The phosphoryl group of ATP is transferred to a nitrogen of
creatine (metabolically derived from the amino acid arginine) to form phosphocreatine.
In creatine, the nitrogen is part of the resonance-stabilized guanidino group; in phospho-
creatine, electrons on the nitrogen are localized. Thus, phosphocreatine is a high-energy
compound. Muscle cells – particularly white (fast) muscle – contain large amounts of the
creatine phosphokinase enzyme, achieving near-equilibrium. During periods of rest, the
reaction runs in the direction of phosphocreatine formation:

(8.26) ATP + creatine ® ADP + phosphocreatine
This direction results from a lowered [ADP] and an increased [creatine] under these condi-
tions. The concentration of ATP is essentially constant. In contracting states, the reverse
reaction occurs:

(8.27) ADP + phosphocreatine ® ATP + creatine
as [ADP] accumulates. The use of a near-equilibrium reaction is illustrated here as the same
reaction can either build up a high-energy phosphate or utilize it to stabilize ATP concentra-
tion. The concentration of phosphocreatine is 40 mM, severalfold that of ATP. ATP itself is
used in the contractile cycle to phosphorylate the “ratchet” protein, myosin. The constancy
of ATP concentration in cells was first demonstrated for muscle itself, which is buffered by
the creatine phosphokinase system. Neither phosphocreatine nor ATP is itself a source of
energy; if the cell were to rely on both, it would sustain its activity for only seconds.
8.9.2 NDP KINASE
A second maintenance reaction for ATP is the nucleotide diphosphate (NDP) kinase, which
catalyzes the reaction:

(8.28) ATP + NDP ADP + NTP
FIGURE 8.7 Creatine phosphokinase mechanism. The mechanism diagram shows the rela-
tively simple reaction pathway that is analogous to a portion of the asparagine synthetase reac-
tion shown in Figure 8.6.
174 8.10 Energy of Redox Reactions
where the nucleotide N is a variable: guanine (G), cytosine (C), or uracil (U). The reaction
allows nucleotides other than ATP to serve as energy donors. The displacement from equi-
librium for the NDP kinase reaction in cells remains unknown, although it is likely to be
metabolically irreversible based on estimates of nucleotide concentrations. In fact, there are
no known instances where this reaction is used to synthesize ATP.
The overall ΔGº for the reaction depicted in Equation 8.28 is approximately zero (and
therefore Keq ≈ 1). This result stems from the fact that the reaction has essentially the same
phosphate bonds in both the reactants and the products: a triphosphate (ATP, NTP) and a
diphosphate (NDP, ADP). It is sometimes difficult to resist the temptation to conclude that
the reaction is near-equilibrium in cells, but there is no basis for this supposition. The other
intriguing aspect of NDP kinase is that it is an enzyme with somewhat relaxed specificities,
accepting different nucleotides as substrate. While this broad substrate selection is true of
some enzymes, most have a highly selective substrate preference.
8.9.3 ADENYLATE KINASE
A third maintenance type reaction for ATP is catalyzed by adenylate kinase:
(8.29) ATP + AMP ADP + ADP
As with the NDP kinase reaction, the equilibrium constant for the adenylate kinase reaction
is approximately 1, its standard free energy is about zero. However, the reaction catalyzed
by adenylate kinase reaction is known to be near-equilibrium in cells. AMP is generated by
reactions that cleave ATP at the α-phosphate position, also producing pyrophosphate. AMP
can be converted to ADP by the adenylate kinase reaction. On the other hand, the accumula-
tion of ADP can lead to AMP formation, which is a cellular signal for certain enzymes (e.g.,
AMP-kinase, Chapter 13).
The enzyme adenylate kinase has binding sites for both ATP and AMP; the two nucleo-
tides attach to the enzyme at the same time (Figure 8.8). Once positioned, the terminal
oxygen of the AMP attacks the terminal phosphoryl of the ATP, resulting in the two ADP
product molecules. The simultaneous presence of both adenine nucleotides is dramati-
cally evident by the potent inhibitor diadenosine pentaphosphate (Figure 8.9), a molecule
consisting of two adenine nucleotides linked with five phosphates. A very similar inhibi-
tor with just one less phosphate, diadenosine tetraphosphate, is an order of magnitude
more potent, providing strong evidence for the precise alignment of the nucleotides on the
enzyme surface.
8.10 ENERGY OF REDOX REACTIONS
We have been introduced to redox reactions in the first chapter. Redox reactions involve a
gain or loss of electrons. A separate description was the direct oxidation by oxygen itself,
useful in rapidly identifying organic molecules in terms of their redox state. A third is the
FIGURE 8.8 Adenylate kinase mechanism. In-line electron flow is made possible by the simul-
taneous binding of ATP and AMP.
FIGURE 8.9 Diadenosine pentaphosphate.
oxidation number, which can be assigned to each atom in a molecule when the loss or gain
of electrons is not as apparent as it is for redox-active ions. In this section, we explore the
energetics of redox transfer.
We begin with a summary of the fundamentals of electricity and introduce the electro-
chemical cell and the standard redox potential. Finally, we will consider the redox poten-
tials of the redox coenzymes we introduced in the previous chapter. Redox coenzymes are
involved in every major pathway in biochemistry.
8.10.1 ELECTRICITY FUNDAMENTALS
The most basic concept of electricity is the charge, which is a primitive, meaning that we
have no deeper understanding; we simply become more comfortable with it as we learn its
features. The rate of flow of a charge with time is the current, measured in amperes. This
unit honors the scientist André-Marie Ampere, who measured the force between two wires
1 m apart. The force results from a magnetic field produced by currents in the wires. A cur-
rent producing a force of 2 × 10−7 newtons (N) in each wire is equal to 1 ampere, or amp. The
newton itself (Box 8.1) is the force required to accelerate 1 kg to 1 m/s2.
Current (I) is a derived electrical term: charge (Q) divided by time (t). Therefore, Q is:
(8.30) Q = I *t
Curiously, while charge is the fundamental unit, its units are derived from the current;
charge is measured in amp-sec or coulombs. The minimum charge – that is, the charge on
one electron – was first obtained by Robert Millikan in his famous “oil drop” experiment of
1910. This experiment involved spraying a fine mist of oil into an electric field and finding the
minimum charge per drop. The value is 1.6 × 10−19 coulombs (C). Combining this number
with the Avogadro number of electrons, we can calculate the number of charges in one mol:
(8.31) (1.6 * 10 -19

)( )
C * 6 * 1023 electrons/mol = 96500 C/mol = F
where F is the Faraday, in honor of Michael Faraday, who discovered electromagnetic

induction.
We have already introduced the concept of energy, with the examples of mechanical and
chemical energies, but all types of energy are interconvertible. Energy interconversion was
implicit in the force on parallel wires and the stabilizing of the oil drops. The unit of energy
in each case is the same: joules (J). The joule is measured in N-m: mechanically, the force of 1
N exerted over a distance of 1 m. An electrical potential, also known as a voltage, is defined
as the energy of separation of charges. The units are volts defined as energy per charge:

(8.32) 1 V = 1 J/C
Voltage is a potential energy, existing so long as there is a separation of charges.

Perhaps the most famous equation in electricity is Ohm’s Law:
(8.33) V = I * R
relating voltage (V) and current (I) to the resistance (R). With voltage and current defined, the
resistance units should be J/C2 but instead are, by custom, taken to be ohms (for yet another
honoree, Georg Simon Ohm). The electrical unit that obeys Ohm’s Law, that is, produces a
linear relationship between V and I is called the resistor, and many materials obey this law.
Voltage is a fundamental parameter of electricity and electronics, used in virtually every
laboratory instrument. However, to analyze redox changes of reactions, the apparatus
requires a voltage across solutions, so we need to consider both types of charge carriers:
electrons in wires and ions in solutions.
8.10.2 THE ELECTROCHEMICAL CELL
The study of electricity itself dates back over 2000 years, but the first demonstration of redox
chemistry was that of Alessandro Volta in 1800. His experiment showed how electrons flow
between copper and zinc metals, creating the first battery (technically, electrochemical
cell).
A bar of Zn metal in a solution containing sulfate counterions (SO42−) will release Zn2+ to
the solution, and leave excess electrons on the Zn bar (Figure 8.10A). For a bar of Cu metal,
the tendency is in the opposite direction; Cu2+ from the solution reacts with electrons in the
bar and plates the metal surface (Figure 8.10B).
Suppose the two metal bars are connected outside the solution by a wire to enable electron
movement, and a salt bridge connects the solutions to allow ion movement. In that case, the
result is the electrochemical cell illustrated in Figure 8.10C. The bars are now considered to
be electrodes. Reactions of electrodes occur at a surface, defining an interface between two
phases. In the cases we are considering, the phases are solid (the bars of metal) and liquid (the
electrolyte solutions). Notice that both types of charge carriers are involved in our analysis:
electrons, carrying charge through solid metals; and solutions, carrying ions through liquids.
FIGURE 8.10 The electrochemical cell. A) A bar of Zn in a solution containing sulfate ions;
Zn2+ tends to come into solution B) A bar of Cu in a solution containing sulfate ions; Cu2+ tends
to move from solution to the surface of the bar C) The electrochemical cell connected by wires
between bars – now electrodes – allows electron flow; the solutions are connected by a salt
bridge, allowing ion flow for a complete circuit. D) A voltmeter inserted into the circuit allows
measurement of the potential.
To measure the process, we insert the leads of a voltmeter into the wire, as in Figure 8.10D.
Since a voltmeter presents a very high resistance to a circuit, there is only a small current
flow through the meter. Under these conditions, the voltage reading reports strictly initial
conditions since there is minimal electron flow in determining the potential. The voltage
measurement is analogous to the measurement of the initial velocity v in enzyme kinetics.
8.10.3 STANDARD REDUCTION POTENTIALS
As is the case with state functions, we can define standards for redox reactions. In the stan-
dard state, all concentrations are 1 M. This will produce a voltage of 1.1 V for the cell depicted
in Figure 8.10D. To compare redox reactions, we need to define a standard reference half-cell.
The agreed-upon reference is the hydrogen electrode system, assigned a potential of zero:
(8.34) H+ + e - ® ½ H 2 ; E 0 = 0 V
Now, if the Zn/Zn2+ half-cell is connected to the hydrogen electrode system, the voltmeter
reports:

(8.35) Zn Zn+2 H+ H 2 ; E0rxn = +0.76 V
and for the Cu2+/Cu half cell
(8.36) Cu Cu 2+ H+ H 2 ; E0rxn = -0.34 V
When we reverse the direction of the Cu/Cu2+ half-cell, making it an oxidation as it is known
to occur, its sign is also inverted so that we obtain the expected result for our original cell:

(8.37) Zn + Cu 2+ Zn 2+ + Cu

(8.38) Zn Zn+2 Cu Cu 2+ ; E0rxn = 1.1 V
E0 values for a wide variety of half-cells are compiled as tables of reduction potentials. It is
somewhat unfortunate that the standard chosen involves the proton, as it is found in so many
biochemical reactions, and its concentration in vivo is on the order of 10−7. Accordingly, the
TABLE 8.4 Reduction Potentials

Half Reaction Eº, Volts
½O2 + 2H + 2e → H2O
+ − +0.81
Fe3+(cyt c) + e− → Fe2+(cyt c) +0.25
Fe3+(cyt b) + e− → Fe2+(cyt b) +0.08
UQ + 2H+ + 2e− → UQH +0.04
fumarate + 2H+ + 2e− → succinate +0.03
pyruvate + 2H+ + 2e− → lactate −0.20
glutathione (S–S) + 2H+ + 2e− + 2H+ + 2e− → glutathione (-SH) −0.23
lipoic acid (S–S) + 2H+ + 2e− → lipoic acid (-SH) −0.29
2H+ + 2e− → H2 −0.42
NAD+ + H+ + 2e− → NADH −0.43
biochemical redox potentials, such as those of Table 8.4, are adjusted to a pH of 7. Note that
the redox potential of H2/H+ with this standard is significantly different from zero.
8.10.4 REDUCTION POTENTIALS AND FREE ENERGY
The conversion of electrical energy represented by E (electromotive force) into chemical free
energy G is direct once we have the Faraday constant, which we developed above. The elec-
trical potential E is energy per charge. The total amount of charge transferred as electrons
is –nF, where n is the stoichiometric number of electrons transferred in the reaction, F is the
Faraday constant. The negative sign is required for electrons:

(8.39) E = energy/charge

(8.40) DG = energy

(8.41) -nF = charge
so that:

(8.42) DG = -nFE
It is a direct substitution to convert the free energy equation into one expressing electrical
potential.

(8.43) DG = DG0 + RT ln Q

(8.44) -nFE = -nFE0 + RT ln Q

(8.45) E = E0 - ( RT/nF ) ln Q
The last equation enclosed in a box is also known as the Nernst Equation. This can be
expressed in the standard state, where Q = 1 and therefore E = E0 so that:
(8.46) E0 = -DG0 /nF
since

(8.20) DG0 = -RT ln K
then

(8.47) E0 = + ( RT/nF ) ln K
We can break out the terms RT/F from the right side of Equation (8.47), assuming the usual
temperature of 25º (298 K), with R = 8.3 J/mol, and F = 96,500 C/mol, and converting ln to
log by multiplying by 2.3:
59 × logK

(8.48) E0 = mV
n
Now we have a means of converting the redox potential to an equilibrium constant. The
term in this equation is for the entire reaction. It is thus possible to interconvert the free
energy (ΔG), the redox potential for the overall reaction (Eº), and the equilibrium constant
(K). Beyond the conceptual advance, electrical measurements provide an alternative means
of measuring an equilibrium constant for a redox reaction without having to establish equi-
librium conditions.
The E values relating to free energy are distinct from the E values for half-reactions. The
former is more properly designated Erxn. The standard free energy for half-reactions is fre-
quently called the midpoint potential. Em. Em is descriptive, since it measures the potential
of the oxidized and reduced form at 1 M and measures them at any value so long as they
are equal in concentration. While it is never the case that the concentrations are at 1 M in
cells, if they are in roughly equal concentrations, then their ratio will be approximately 1,
so that it is immaterial what their actual concentrations are. This point helps us understand
the redox positions of electron transfer species in the mitochondrial respiratory chain in
Chapter 11. However, it is also of interest in understanding the redox potentials of bound
cofactors in general. In particular, the flavin cofactors are bound. Thus, to a first approxima-
tion, the actual midpoint potential of FAD/FADH2 for any given reaction is the same as its
standard reduction potential.
8.10.5 REDUCTION POTENTIALS
Table 8.4 provides a list of reduction potentials. They are ordered from most positive to most
negative. There are several points that we can appreciate from this listing. First, we notice that
the top entry, the reduction of oxygen to water, has the highest listed potential. The relation-
ship between Eº for an overall reaction and ΔGº is inverse: a large positive midpoint potential
means a large negative standard free energy. Thus, under standard conditions, the table is
ordered in terms of exergonic to more endergonic. Notice the standard potential of protons
to H2 is −0.42; this would be zero if [H+] was 1 M. It only matters that the values are relative,
but it is clear that it becomes useful to have this pH value set at the more physiological level.
The midpoint potential of NAD+/NADH is large and negative; thus, under standard condi-
tions, the reverse reaction is favored. A further point should be noted: in the two examples
where Fe3+ is reduced to Fe2+, the values of Eº are quite different; these are the same ions. The
reason is that they are in a different environment. Thus, the prediction of redox potentials
from the species itself can be misleading. A significant influence on the midpoint potentials
is the electrical environment of the molecules. For example, cytochromes are associated with
the mitochondrial membrane, which has a membrane potential across it (see Chapter 11).
A further point is that standard redox potentials may be very different from actual poten-
tials for mobile cofactors. While the standard potential for NAD+/NADH is not very differ-
ent from the midpoint potential within the mitochondria, it is far more oxidized in the cell
cytosol.
A table of redox potentials makes it a simple matter to predict which reactions are at least
theoretically possible. Since we now know that an overall Eºrxn must be positive for the ΔGº to
be negative, it is a simple matter to arrange a possible redox reaction between two half-reac-
tions listed in Table 8.4 and determine if the reaction is possible. To take an extreme example,
180 8.11 Mobile Cofactors and the Pathway View
consider the possibility that NAD+/NADH pair reacts with the O2/H2O pair. We need only
add the appropriate midpoint potentials. The oxygen-containing half-reaction occurs in the
direction presented, as it is large and positive. The other half of a redox reaction, NAD/
NADH, must proceed in the reverse direction. To perform the calculation, we write:
Eº
Reaction mV
½O2 + 2H+ + 2e− → H2O +0.81
NADH → NAD+ + H+ + 2e− +0.43
net ½O2 + 2H+ + NADH → H2O + NAD+ + H+ +1.24
This is a thermodynamic calculation, not a kinetic mechanism. There is no reaction that

directly connects these two half reactions in biology. There is an important indirect route,
involving mitochondria as presented in Chapter 11. However, this analysis tells us if the over-
all reaction is thermodynamically possible. Even with a small table of Reduction Potentials,
many such theoretical calculations can be performed. The power of thermodynamics is not
that it can guarantee which reactions happen; it is a reliable test of which ones are possible
and which are not possible.
8.11 MOBILE COFACTORS AND THE PATHWAY VIEW
As presented in Chapter 7, the intermediates ATP, ADP, NADH, and NAD+ – as well as
closely related intermediates such as GTP, GDP, NADPH, and NADP+ – are mobile cofac-
tors when they are viewed in the context of metabolic pathways. Mobile cofactors are links
between or within metabolic sequences. Connections between reactions that occur in a
pathway view are not present in isolated reactions. The most fundamental connection – the
one that defines a pathway – is that the product of one reaction is the substrate of the next.
Consider the pathway shown in Figure 8.11. One of the interior reactions uses ATP as sub-
strate and forms ADP as product. From the standpoint of the pathway, ATP and ADP are not
substrates or products; they are mobile cofactors. Unlike the pathway intermediates involved
FIGURE 8.11 ATP and ADP as pathway cofactors. In the pathway on the left, the ATP and ADP
are mobile cofactors. In the expansion on the right, highlighting the reaction of S2 → S3, they are
substrate and product, respectively.
FIGURE 8.12 NAD+ and NADH connect pathways. The pathway on the left involving an inter-
mediates reduces NAD+. The pathway on the right, involving bn intermediates and oxidizes NADH.
in this reaction, ATP and ADP are not directly connected to either the previous or the fol-
lowing reactions, but rather to reactions elsewhere in this or another pathway.
The other mobile cofactors, such as NADH and NAD+, act in the same way; namely, they
connect different pathways in parallel, as suggested by Figure 8.12. This behavior is very
different from cofactors that always stay bound to their enzymes. The chemistry of bound
cofactors – often called prosthetic groups – may resemble mobile cofactors. However, pros-
thetic groups are attached to the enzyme and must be regenerated along with the rest of the
enzyme in the course of a single catalytic cycle (Chapter 7). Thus, they cannot connect sepa-
rate pathways. If we consider the first substrate for a pathway as the pathway substrate and
the last product as the pathway product, then all of the other intermediates of the pathway
reach a constant concentration once the pathway has achieved a steady-state. The steady-
state for the intermediates is maintained by a constant provision of pathway substrate and
constant removal of pathway product. However, the mobile cofactors are in limited supply, so
they must be regenerated by a separate pathway in sufficient amounts to allow the pathway to
proceed. Glycolysis exemplifies cofactor balance using both ATP and NADH.
SUMMARY
Energy exchanges are the central issue of metabolism. The major energy intermediate is the
molecule ATP. A deeper understanding of energy requires a study of thermodynamics, which
has two key laws. The first law states that the total internal energy of a system is conserved
between heat (q) and work (w). Internal energy is itself a state function, defined as (q–w). The
second law involves entropy, which is roughly a measure of “disorder”, but in actuality is a
measure of the possible number of states of a system and their arrangement. According to
the second law, the entropy of the universe always increases after a reaction. The free energy
combines the internal energy and the entropy of the system to produce a parameter, ΔG,
which determines whether the reaction can take place as written. If ΔG is negative, then the
reaction is exergonic and can take place. If ΔG is positive, then the reaction is endergonic,
and the reverse reaction can take place. If ΔG = 0, then the reaction is at equilibrium. Under
standard conditions, all reactions have substrate and product concentrations of 1M and can
be compared to one another in vitro. Under cellular conditions, the reactions are displaced
from equilibrium, either slightly – in which case the reaction is near-equilibrium – or greatly,
in which case the reaction is metabolically irreversible. Redox reactions involving gain or loss
of electrons can be analyzed by separating the oxidation from the reduction half-reactions.
The separation can be performed experimentally in some cases in an electrochemical cell.
A reduction potential characterizes redox reactions; half-reactions are arranged in order of
their ability to become reduced. There is a direct relationship between the redox potential
and the standard free energy. Two energy molecules, ATP and NADH, represent mobile
cofactors that connect separate pathways in metabolism. ATP is involved in several reactions
that exchange high-energy phosphate: creatine phosphokinase, in which a separate store of
high-energy phosphate can be produced; NDP kinase, which exchanges phosphoryl groups
with nucleotides other than adenine; and adenylate kinase, which reversibly interconnects
ATP, ADP, and AMP. Mobile cofactors connect reaction sequences – pathways – in parallel.
REVIEW QUESTIONS
1. A reaction has an enthalpy change of 30 kJ/mol and an entropy change of 100J/molK.

Assuming room temperature is about 300 K, is this reaction at equilibrium? What
would the free energy change be if the temperature was higher?
2. Suppose that a reaction has ΔG0 = 0. What conclusions can be drawn about its equi-
librium constant? Can we determine if it is reversible under cellular conditions?
3. Internal energy is very similar to enthalpy. Explain why that is the case biologically
and why we still retain enthalpy in equations for free energy.
4. Redox reactions often involve the transfer of hydrogens during reactions involving
organic compounds; acid–base reactions do as well. How are these processes distinct,
and why do both seem to involve hydrogen transfer?
5. What is the most reduced form of carbon in an organic molecule? What is the most
oxidized?
6. Explain why heat is not a state function, but heat at constant pressure is a state
function.
7. Perfect interconversion between heat and work does not violate the first law of ther-
modynamics. Why does it violate the second law of thermodynamics?
8. One way of calculating an equilibrium constant is to use standard free energy values.
Here, Equation 8.20 has been modified to use common (base 10) logs:
DGo = - ( 2.3 ) RT log K eq
Use this equation to calculate Keq, if ΔGº is (a) 1; (b) 100; (c) 1/100.
9. Because heat is actually just an energy exchange due to a temperature difference,
what does it mean when you open the window in winter and the cold air comes in? Is
heat, instead, really flowing out?
10. The redox potential for NAD+/NADH is very large and negative. How is it that any
reaction could cause a reduction of NAD+?
CHAPTER 8 ADDENDUM: FREE ENERGY DERIVATION
We have asserted that there is a function that combines the first and second laws of thermo-
dynamics and named this free energy. Let us return to the heat transfer (q) under constant
pressure to get a sense of its origin. This term is symbolized alternatively as qP or ΔH. Making
a slight change to ΔH (which is redundant but important for our purpose here), let us call it
ΔHsys, to emphasize that this is a system variable.
Our sign convention defines the direction of q as positive when heat is transferred to the
system. Now let us suppose we are interested in the heat transferred to the surroundings.
Since qP is a system variable, then the heat transferred to the surroundings must have the
same value, but the opposite sign:

(CA8.1) q surr = -q P
If we divide both sides by the temperature T:
q surr q

(CA8.2) =- P
T T
The term on the left-hand side of Equation (CA8.2) is the entropy of the surroundings, and
thus:
q surr q

(CA8.3) DSsurr = =- P
T T
The second law of thermodynamics is the statement of the entropy change of the universe
(for any real process) is greater than zero. As the universe is itself the sum of the system and
surroundings, then:
(CA8.4) DSuniv = DSsurr + DSsys > 0
Substituting Equation (CA8.3) into Equation (CA8.4):
qP

(CA8.4) - + DSsys > 0
T
or equivalently:
DH sys

(CA8.4) - + DSsys > 0
T
If we multiply first by T and then by −1, we obtain:

(CA8.5) DH sys - TDSsys < 0
In the process of multiplying Equation CA8.4 by −1, the inequality sign inverts, so the equa-
tion now states that the terms on the left-hand side of Equation CA8.5 are less than zero.
This is essentially the entire derivation; all that is left is to define the left-hand side as the free
energy and assign it a symbol, ΔG:

(CA8.6) DGsys = DH sys - TDSsys < 0
KEY TERMS
amperes
charge
coulombs
current
electrical potential
electrochemical cell
electrodes
184 Bibliography
endothermic
energy coupling
entropy
exergonic
exothermic
Faraday
free energy
heat
internal energy
mass action ratio
metabolically irreversible
midpoint potential
near-equilibrium
Nernst Equation
nonstate
ohms
oxidation number
path-dependent
path-independent
primitive
PV work
standard conditions
state
state variables
system
temperature
thermodynamics
voltage
work
BIBLIOGRAPHY
R.A. Alberty. Standard Apparent Reduction Potentials of Biochemical Half Reactions and
Thermodynamic Data on the Species Involved. Biophys. Chem. 111 (2004) 115–122.
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Glycolysis 9
Glycolysis was first discovered as an activity in yeast known as fermentation (see Box 9.1). In
mammals, it is present in every cell. Every enzymatic step of glycolysis has been thoroughly
studied, and thus the pathway can be considered a prototype of other metabolic routes. Both
glycolysis and the Krebs Cycle (Chapter 10) can be regarded as the crossroads of metabolism.
In each, every intermediate is connected to another pathway in most cells.
A key purpose of glycolysis is the generation of the energy intermediate ATP. The sequence
from extracellular glucose to lactate is outlined in Figure 9.1. The intermediates on the top
portion are 6-carbon molecules. After a splitting reaction, the intermediates are 3-carbon
molecules, shown in the lower portion of the figure. Thus, the flux of the lower portion is
double that of the upper. The overall reaction of glycolysis is:
(9.1) glucose + 2 ADP + 2 Pi ® 2 lactate + 2 ATP
showing the mobile cofactors ADP, Pi, and ATP but not NAD and NADH, even though the
latter two are involved in the overall sequence.
In this chapter, we will first examine each step of the pathway separately, presenting an
indication of the reaction mechanism, and regulatory features. We will then explore other
pathways that intersect with glycolysis.
9.1 GLUCOSE TRANSPORT
Before glucose can be converted to pyruvate, it must first enter the cell. All of the enzymes
catalyzing the steps of glycolysis are present in the cytosol. While some cells use a special-
ized form of glucose uptake (Box 9.2), most mammalian cells use a passive glucose transport
protein, abbreviated GLUT. There are 14 GLUT isoforms – that is, distinct proteins with a
similar function. These are named in chronological order of their discovery (e.g., GLUT1,
GLUT2, etc.). We will examine three of them here (Table 9.1).
GLUT1, first discovered in red blood cells, is also found in many mammalian cells and
is prominent in fetal tissues. A relatively low-K M transporter (about 2 mM; for comparison,
blood glucose concentration is 5 mM) provides baseline glucose uptake for most cells. The
protein spans the membrane with 12 alpha-helical segments and adopts a clamshell-like con-
figuration. Glucose binding on one side induces a conformational change that flips the orien-
tation of its binding site to the other side (Figure 9.2). The slow step of the mechanism is the
return of the unoccupied receptor to the original membrane face. Like all GLUT transport-
ers, glucose can traverse the membrane in either direction. However, GLUT1 is unidirec-
tional, a condition enforced in most cells that maintain a relatively low intracellular glucose
concentration of about 0.1 mM. In terms of our thermodynamic characterization developed
in Chapter 8, we would consider GLUT1 a metabolically irreversible reaction.
GLUT2 is found in the liver, pancreas, kidney, and intestinal epithelia. The transporter
operates well below saturation; its KM is above 10 mM. GLUT2 is unique among the GLUT
proteins as it achieves near-equilibrium with extracellular glucose concentrations. In cells
with a GLUT2 isoform, the intracellular glucose concentration is similar to that of extracel-
lular glucose (about 5 mM). Thus, glucose uptake through GLUT2 is very sensitive to changes
185
186 9.2 From Glucose to Pyruvate
Box 9.1 Word Origins: Zymo
Zymo and its variants are from Greek, meaning leaven. The root term refers to yeast and
to ferments from yeast. When Eduard Buchner found that extracts derived from yeast
degrade glucose in a cell-free fermentation, he applied the name enzyme to their prepa-
ration. Similarly, there are fungal diseases called zymotic, and the sign of infection was
named zymosis, which means ferment. The yeast extract that metabolizes glucose was
later discovered to be a mixture of many components. Each of those (and thousands
more) is now called an enzyme. Thus, glycolysis became the first process recognized to
act independently of a cell. Embedded in the name “enzymes” are the yeasts, the organ-
isms in which the first collection of enzymes producing an observable result – namely,
bubbles – was discovered.
FIGURE 9.1 Glycolysis. The overall pathway of glycolysis is presented with the mobile cofac-
tors outlined in blue. Abbreviations for each of the intermediates between glucose and pyruvate
are also shown.
in extracellular glucose concentrations, and the transporter is also used for the export of cel-
lular glucose by other pathways.
GLUT4 is found in muscle and fat cells. Like GLUT1, it is metabolically irreversible and
has a relatively low K M for glucose. The unique feature of GLUT4 is insulin sensitivity, provid-
ing an increased uptake of glucose in response to an increase in blood insulin concentration.
The GLUT4 protein is believed to translocate from an internal cell membrane to the plasma
membrane in response to insulin.
9.2 FROM GLUCOSE TO PYRUVATE
9.2.1 HEXOKINASE
The first step of glycolysis from intracellular glucose is catalyzed by hexokinase. The reaction
is a transfer of the phosphoryl group of ATP to glucose:

(9.2) Glucose + ATP ® Glucose-6-P + ADP
As in all kinase reactions, the true substrate is MgATP, although the presence of the metal
is usually not indicated. The mechanism (Figure 9.3) is typical of kinase reactions, such as
the adenylate kinase reaction (Chapter 8). As indicated in the figure, the nucleophile – in
Chapter 9 – Glycolysis 187
Box 9.2 A Separate Class of Glucose Transporters
The glucose transporters of the GLUT class described in the text are the most commonly
used means of glucose traversing membranes. In certain epithelial cells – that is, those
with distinct membranes facing different spaces – there is another type of transporter
that catalyzes the uptake of extracellular glucose along with extracellular Na+. These
transporters are indirectly energy-linked, as ATP is expended in maintaining the sodium
gradient: high outside the cell and low inside. Thus, as Na+ flows down its gradient, glu-
cose enters along with it in the presence of a sodium-linked glucose transporter (SGLT).
For example, the SGLT in the intestine allows the entry of glucose from the lumen into the
cell. Little of the glucose is actually metabolized in the epithelial lining of the intestine;
instead, most is transported out of the cell into the blood to be carried to other cells by
virtue of having a GLUT2 in the basolateral membrane (Figure B9.2). Another SGLT iso-
form exists in the kidneys, allowing glucose filtered into tubules to be transported into
the tubule cell across an apical membrane, and transported out again using a GLUT2 in
the basolateral membrane for reabsorption into the blood.
FIGURE B9.2 Glucose transport in intestinal mucosa. Two glucose transporters are present
in the lining cells of the small intestine. SGLT catalyzes the co-transport of glucose and Na+
across the mucosal membrane, symbolized in the figure as having microvilli that expand the
surface area for absorption. Glucose exits the cell through the GLUT2 in the basolateral mem-
brane, which leads glucose into the blood capillary and thus the circulatory system.
TABLE 9.1 Properties of Some Glucose Transporters

Transporter Cell Types Near-Equilibrium Insulin Sensitive KM for Glucose, mM
GLUT1 Red blood cells, many others no no 2
GLUT2 Liver, pancreas yes no > 10
GLUT4 Muscle, fat no yes 1
this case, the 6-hydroxyl group of glucose – initiates a nucleophilic attack on the partially
positive phosphorous of the terminal (γ) phosphate of ATP. Electrons of the π-bond migrate
to the oxygen atom, forming a pentavalent phosphate intermediate. As these electrons move
back from the oxygen, they displace electrons of the P–O bond, as shown, releasing ADP.
While any of the P–O bond electrons may move, the others would result in a reversion
to the substrate. There are hundreds of kinase reactions in the cell, and they all have this
mechanism.
Another feature of hexokinase (in common with other kinases) is the protection of
the intermediates from hydrolysis. A conformational change in the enzyme upon bind-
ing the substrate isolates the active site and thus protects it from spurious water molecules
(Figure 9.4). Note the reappearance of the “clamshell” configuration also apparent in the
FIGURE 9.2 Glucose transport. The transporter undergoes a conformational change when
glucose is bound. Glucose binding to the exterior causes the face of the transporter to flip to the
interior, where glucose can dissociate and enter the cytosol. The slow step is the flipping of the
unoccupied transporter to face the exterior once again.
FIGURE 9.3 Mechanism of hexokinase. Simultaneous binding of ATP and glucose to the
enzyme provides the proximity for the nucleophilic attack by the 6-hydroxyl group of glucose
on the terminal phosphoryl of ATP. Electron rearrangement leads to the production of glucose-
6-P and ADP.
FIGURE 9.4 Clamshell model of hexokinase. After binding substrates, the reaction takes
place in the interior of the enzyme, which is hydrophobic and excludes water, preventing ATP
hydrolysis.
GLUT protein (Figure 9.2). In both cases, the two domains comprising the clamshell are
connected by a hinge region.
Just as there are isoforms of the glucose transporter, different proteins, called isozymes,
all catalyze the hexokinase reaction. Most hexokinase isozymes have a similar affinity for
glucose, estimated by their K M values – about 1 mM. One isozyme has an unusually high K M
for glucose, and is named glucokinase. The same cells that have glucokinase also have the
GLUT2 transporter. Thus, like GLUT2, glucokinase is found in the liver and the endocrine
cells of the pancreas. This pairing has a purpose: because GLUT2 allows a near-equilibrium
transport of glucose across the membrane, the intracellular glucose concentration will be
very high – near the blood glucose value of 5 mM. Accordingly, the K M for glucokinase is also
very high, about 20 mM. Unlike GLUT2, glucokinase is a metabolically irreversible reaction,
like all hexokinase isozymes.
Aside from glucokinase, the other hexokinase isozymes are product-inhibited by glu-
cose-6-P (i.e., the product of the hexokinase reaction). Therefore in most cells, inhibition of
subsequent reactions of glycolysis leads to the accumulation of glucose-6-P and hexokinase
inhibition. Once formed, glucose-6-P is “trapped” in the cytosol. This confinement is not
because of the greater water solubility of the phosphorylated compound but rather because
this phosphate ester – like all of the subsequent intermediates of glycolysis up to pyruvate
formation – has no transporter that would enable exit from the cell. The composition of
every water space in the cell depends on the presence of transporters that allow them to enter
and exit.
9.2.2 GLUCOSE PHOSPHATE ISOMERASE
The next step in glycolysis is the isomerization of glucose-6-P to fructose-6-P. The enzyme
glucose-6-P isomerase catalyzes this near-equilibrium reaction, converting an aldehyde to a
ketone sugar phosphate. Figure 9.5 shows that the reaction requires a nonenzymatic opening
of the ring form of glucose-6-P to reveal the aldehyde.
The mechanism of glucose-P isomerase is illustrated in Figure 9.6. The reaction flow is
drawn above the dotted line, and intermediates involving electron rearrangements are below
the dotted line. An attached base (B) abstracts a proton from the C2 position. The subsequent
electron rearrangement leads to an intermediate enediol, a symmetrical intermediate shown
in the center of Figure 9.6. Note the addition and loss of protons on the oxygen atoms of the
enediol. These reactions are exchanges with water molecules and occur readily. The middle
FIGURE 9.5 Glucose phosphate isomerase ring opening. Glucose phosphate must achieve
the open-chain form before it can be isomerized to fructose phosphate. These reactions are
nonenzymatic and do not affect the rate of the enzymatic reaction.
FIGURE 9.6 Mechanism of glucose phosphate isomerase. Essentially a simple acid-base-

catalyzed reaction, a proton is first abstracted from C2 of glucose-6-P by an enzyme-bound
basic group, and electron rearrangement produces the symmetrical intermediate (the enediol).
The proton is added back to the enediol but at C1. Electron rearrangement then produces
fructose-6-P.
reaction above the dotted line of Figure 9.6 indicates this exchange. In the remainder of the
mechanism, the protonated enzyme-linked base becomes positioned near Cl, and donates
the proton to Cl as a result of electron movement initiated from electrons on the oxygen
attached to C2. Both the intermediate and the mechanism are virtually symmetrical, a fea-
ture that we will see repeated with other isomerase-type enzymes.
9.2.3 PHOSPHOFRUCTOKINASE
Phosphofructokinase (PFK) catalyzes the phosphorylation of fructose-6-P to fructose-1,6-P2

(Figure 9.7). This phosphoryl transferase reaction is virtually identical in mechanism to the
hexokinase reaction. PFK is metabolically irreversible and the major rate-limiting step in
glycolysis.
PFK is regulated allosterically by a large number of molecules in vitro (Box 9.3). Two allo-
steric regulators are physiologically significant: citrate (a negative modulator) and fructose-
2,6-P2 (a positive modulator). Both effects on the enzyme activity are indicated in Figure 9.8
as alterations in the S-shaped substrate-velocity curve.
Citrate regulation is a key part of the Pasteur effect, a 19th-century observation that glu-
cose utilization is decreased in the presence of oxygen. Citrate is an intermediate in the Krebs
FIGURE 9.7 Phosphofructokinase reaction.
FIGURE 9.8 Allosteric regulation of phosphofructokinase. In the presence of fructose-2,6-P2,

the curve shifts to the left, indicating activation. In the presence of citrate, the curve shifts to the
right, indicating inhibition.
Box 9.3 In Vitro Modulators of PFK
Beyond fructose-2,6-P and citrate, a large number of other allosteric effectors of PFK are
known in vitro. However, they are unlikely to be physiological regulators of the enzyme
in the cell. An old observation is that ATP inhibits PFK as well as many other reactions
in vitro. But the fact that the concentration of ATP is nearly constant in cells means that
this energy intermediate does not serve as a cellular regulator. Other candidates can be
excluded for different reasons. For example, ADP activates PFK in vitro and does change
in concentration, but its cellular concentration is orders of magnitude lower than that
needed to activate the enzyme in vivo. As an additional example, the concentration of
fructose-1,6-P2 does change in cells, is known to be a feed-forward activator of pyruvate
kinase, and was once believed also to be a product activator of PFK. However, fructose-
1,6-P2 only mimics the actual PFK activator, fructose-2,6-P2, which exists at even lower
concentrations.
cycle (Chapter 10), and an increase in its concentration inhibits phosphofructokinase and,
consequently, glycolysis. This regulatory feature adjusts for the fact that oxidative metabo-
lism is far more efficient than glycolysis in energy production.
Fructose-2,6-P2 is formed from fructose-6-P via a reaction catalyzed by PFK-2
(Figure 9.9). This enzymatic reaction is chemically similar to phosphofructokinase.
This figure also shows a separate enzymatic reaction that catalyzes the dephosphory-
lation of fructose-2,6-P2 , forming fructose-6-P. The two enzymatic reactions shown in
Figure 9.9 are physically joined into a single protein that is a kinase and a phosphatase.
Only one enzymatic activity prevails for a given physiological condition. For example,
increased glucagon in the blood leads to the activation of the phosphatase activity in
liver cells. The result is that fructose-2,6-P2 is dephosphorylated, and the activity of PFK
FIGURE 9.9 Phosphofructokinase-2. The enzyme PFK-2 is multifunctional. It catalyzes the

phosphorylation of fructose-6-P to fructose-2,6-P2, and a separate active site of the same protein
catalyzes the dephosphorylation of fructose-2,6-P2 to fructose-6-P. Only one of these processes
is active at any given time. PFK-2 controls the cellular level of fructose-2,6-P2 and, consequently,
the activity of PFK-1 (phosphofructokinase).
FIGURE 9.10 The fructose-2,6-P2 bypass. The two reactions catalyzed by PFK-2 are shown in
the context of the glycolytic pathway. Note that F6P is the substrate of two reactions (PFK-2
and aldolase), as well as the product of two reactions (PFK-2 phosphatase activity and glucose-P
isomerase).
and, subsequently, glycolysis is decreased under these conditions. As a further example,

cancer cells, which have a high glycolytic rate, have an activated PFK-2 kinase and a high
concentration of fructose-2,6-P2 .
The formation of fructose-2,6-P2 can be considered a bypass of the glycolytic pathway.
With the route from glucose to pyruvate outlined in abbreviated form, that bypass is illus-
trated in Figure 9.10. It is evident that F6P (fructose 6-P) has two possible routes: through
PFK-1, to form FBP (fructose–1,6–P2), or through PFK-2, to form fructose-2,6-P2. The latter
serves as a regulator rather than an intermediate and, therefore, does not represent a major
loss of carbon from glycolysis.
9.2.4 ALDOLASE
Aldolase catalyzes the reaction:

(9.3) Fructose-1, 6-P2 Dihydroxyacetone-P + Glyceraldehyde-P
which is at near equilibrium in cells. As it converts a six-carbon to two three-carbon inter-

mediates, this step is at the crossroads between these two stages of glycolysis.
In the aldolase mechanism, a Schiff base (See Chapter 7 and Figure 7.17) forms between
the carbonyl of the substrate and a lysine residue of the enzyme. An outline of the mecha-
nism is shown in Figure 9.11. A splitting of the fructose-1,6-P2 produces one product, glycer-
aldehyde-3-P (GAP), with the remaining fragment still covalently bound to the enzyme in a
Schiff base. Hydrolysis of the Schiff base produces the second product, dihydroxyacetone-P.
The details of the splitting reaction are presented in Figure 9.12. A basic amino acid resi-
due of aldolase attacks the proton from the hydroxyl group at C4, and electron rearrange-
ment leads to breaking the bond between C3 and C4. GAP is released from the enzyme,
leaving the covalently bound intermediate shown in Figure 9.12(b). The intermediate has the
FIGURE 9.11 Aldolase. Aldolase (E in the figure) forms a Schiff base with the substrate, releas-
ing the first product (glyceraldehyde-3-P) with the other three-carbon fragment still bound as a
Schiff base. Reversal of Schiff base formation releases the second product, dihydroxyacetone-P.
FIGURE 9.12 Mechanism of aldolase. A basic residue of the enzyme removes a proton that
leads to the electron rearrangement shown in (a). The splitting is favored because of the reso-
nance stabilization shown as (b) and (c). The subsequent formation of DHAP is due to a reversal
of the Schiff base.
resonance form shown in Figure 9.12(c). The resonance stability contributes to the bond scis-
sion in the first step. The remainder of the mechanism is just the reversal of Schiff base for-
mation, regenerating the free enzyme and forming the second substrate, dihydroxyacetone-P
(DHAP).
9.2.5 TRIOSE PHOSPHATE ISOMERASE
A near-equilibrium interconversion between glyceraldehyde-P and dihydroxyacetone-P, the

two triose phosphates formed by the aldolase step, is catalyzed by triose phosphate isomer-
ase. We have already examined the mechanism for moving a carbonyl group from one car-
bon to its neighbor in the glucose-6-P isomerase reaction above. The very same mechanism
applies to the triose phosphate interconversion:

(9.4) Glyceraldehyde-P Dihydroxyacetone-P
The same base abstractions and enediol intermediate occur, so no new mechanistic details
need to be considered.
All subsequent reactions of glycolysis proceed from glyceraldehyde-P. This means that
triose-P isomerase acts to convert all the dihydroxyacetone-P produced at the aldolase step
into glyceraldehyde-P. Thus, after this enzymatic step, glycolysis has achieved the conversion
of each glucose molecule into two molecules of glyceraldehyde-P.
9.2.6 GLYCERALDEHYDE PHOSPHATE DEHYDROGENASE
The first enzyme mechanism ever solved was that of glyceraldehyde phosphate DH. This
enzyme is also unique, as it catalyzes the only oxidation reaction in glycolysis. Overall, the
reaction converts glyceraldehyde-P to 1,3–bis-P-glycerate, with the concomitant reduction
of NAD+ to NADH (Figure 9.13). In addition to NAD+ and NADH, a third mobile cofactor –
inorganic phosphate (Pi) – is involved in the reaction. The reaction is near-equilibrium in
cells, so it is not involved in cellular regulation.
The mechanism for this reaction (Figure 9.14) shows the participation of the cysteinyl -SH
group in the reaction. The sulfur atom of cysteine serves as a nucleophile that attacks the
aldehyde of the substrate while an enzymatic base residue simultaneously removes the pro-
ton (i.e., a concerted reaction). Next, the enzyme binds NAD+, and an electron relay ensues
between the substrate and the NAD+, terminating at the positively charged nitrogen, an elec-
tron sink. Overall, this step is a hydride transfer (i.e., the movement of hydrogen contain-
ing a pair of electrons) to NAD+. The C1 of the triose phosphate is oxidized, while NAD+ is
reduced to NADH. Finally, an inorganic phosphate binds, displacing the thioester in another
nucleophilic substitution, producing the product 1,3-bis-P2-glycerate, and regenerating the
original form of the enzyme.
The same -SH group that participates in the glyceraldehyde-P DH catalytic mechanism
can react with heavy metals, such as lead and other electron-deficient species, leading to
enzyme inhibition. However, as there are many other -SH groups in most cells, it is a rela-
tively nonselective site of inhibition.
FIGURE 9.13 Glyceraldehyde phosphate DH reaction.

FIGURE 9.14 Mechanism of glyceraldehyde phosphate DH. An enzyme base (B:) removes a
proton from an enzyme-linked -SH group; simultaneously, electrons from the S-H bond attack
the C1 of glyceraldehyde-P; this is a concerted attack. In the second step, the enzyme-bound
glyceraldehyde-P reacts with NAD+, which is positioned to remove a hydride from the sub-
strate and form NADH, which then dissociates. Finally, Pi displaces the bound substrate from the
enzyme, yielding 1,3-bis-P-glycerate.
FIGURE 9.15 The P-glycerate kinase reaction
9.2.7 PHOSPHOGLYCERATE KINASE
The reaction catalyzed by phosphoglycerate kinase (abbreviated P-glycerate kinase) is the

first step of glycolysis in which ATP is produced (Figure 9.15). The reaction is near-equilib-
rium, and its mechanism is the same as other the kinases, such as the hexokinase and phos-
phofructokinase we considered earlier. Kinase reactions are common in biochemistry – they
number in the hundreds – and they all follow the same mechanism. While most kinase reac-
tions are metabolically irreversible, this conclusion can only be drawn after experimental
measurement and not merely because it is energy-linked. It bears repeating that the standard
free energy change of a reaction does not, in general, predict its energy status in living cells.
9.2.8 PHOSPHOGLYCERATE MUTASE
Mutase reactions are phosphoryl transfers that produce an apparent movement of a phos-
phoryl group from one part of the molecule to another. These are typically near-equilibrium
reactions in cells, as is the case for phosphoglycerate mutate (Figure 9.16).
The mechanism for the enzyme involves a phosphorylated histidyl residue (Figure 9.17).
Upon binding to the substrate, the hydroxyl group in the 2-position attacks the histidyl-
phosphate. The resulting intermediate is 2,3-bis-P2-glycerate. Next, the histidyl residue
of the enzyme attacks the phosphoryl in the 3-position of the intermediate compound,
FIGURE 9.16 The P-glyceromutase reaction.
FIGURE 9.17 Mechanism of P-glyceromutase. The free enzyme has a phospho-histidine resi-
due, which is subjected to nucleophilic attack by the substrate hydroxyl group at C2. Removing
the phosphoryl group from the enzyme produces the 2,3-bis-P-glycerate intermediate. In
the second phase, the histidine serves as nucleophile, attacking the phosphoryl group at C3.
Subsequently, the product, 2-P-glycerate, is released, and the original enzyme form is restored.
reforming the histidyl-phosphate enzyme (the original enzyme form) as well as the product,
2-P-glycerate. There is a symmetry to this reaction, bearing a similarity to glucose phosphate
isomerase described above. In both cases, a symmetrical intermediate is created. The enediol
of Figure 9.6 and the 2,3-bis-P-glycerate of Figure 9.17 are drawn side by side in Figure 9.18.
Both mechanisms are also symmetrical; reactions leading to and following the symmetrical
intermediate are mirror images of one another.
9.2.9 ENOLASE
Enolase catalyzes a near-equilibrium reaction converting 2-P-glycerate to P-enolpyruvate

(PEP), with an intermediate anion as illustrated in Figure 9.19. The mechanism is strictly an
acid-base catalyzed one, with one unusual feature: the carboxylate group and its intermedi-
ate anion form are chelated to a pair of Mg2+ ions. The metal ions themselves are attached
to the enzyme through chelate bonds to several amino acid residues as well as to one of the
oxygens of the phosphate group.
In the overall reaction, the substrate undergoes loss of the alkyl hydrogen, colored in red,
and the alkyl hydroxyl group, colored in blue (Figure 9.19). Nonenzymatic removal is not pos-
sible as the alkyl hydrogen has a pKa of about 30, and the C3 hydroxyl is a very poor leaving
group.
The mechanism of Figure 9.20 shows how these reactions take place in the enolase mech-
anism. Panel (a) shows the oxygen atoms of the substrate chelated to the two Mg2+ ions.
A lysine residue serves as a base that abstracts the proton (colored in red), enabled by the
concerted electron rearrangement indicated in this panel. The two Mg2+ ions stabilize the
resulting anion form of the carboxylate shown in panel (b). The acid catalysis is provided by
FIGURE 9.18 Symmetrical intermediates: enediol and vicinyl phosphates. The general nature
of isomerase reaction intermediates is a symmetrical intermediate. Shown here are those of the
Glucose-P-isomerase reaction (a) and the P-glyceromutase reaction (b).
FIGURE 9.19 The enolase reaction.
FIGURE 9.20 Mechanism of enolase. The bound pair of Mg2+ ions is chelated to the oxygen
atoms of the substrate and to several residues of the enzyme. The chelate interaction with the
enzyme stabilizes the electron flow in (a) – initiated by the lysine (base) extraction of proton – to
produce the anion intermediate (b). Next, a glutamate residue protonates the hydroxyl group,
creating an electron sink as well as a leaving group (water). The ensuing electron flow shown in
(c) restores the carboxylate, water is removed, and the product PEP formed (d).
a glutamate residue, as shown in panel (b) that produces the protonated hydroxyl group of
panel (c), an excellent leaving group. The electron rearrangement shown in panel (c) shows
how the restoration of the carboxylate group and water removal is accomplished.
Enolase is known as a site of inhibition by fluoride ions. While the inhibition is only mod-
estly potent and not selective (fluoride ions inhibit other enzymes), there are few other inhib-
itors of glycolysis. Fluoride inhibits enolase by forming chelate bonds to one of the Mg2+ ions
of the enzyme.
9.2.10 PYRUVATE KINASE
The pyruvate kinase step (Figure 9.21) is the second of the ATP-forming reactions of gly-
colysis. Its mechanism is unremarkable because it is the same as the other kinases we have
FIGURE 9.21 The pyruvate kinase reaction.
FIGURE 9.22 Enol-pyruvate intermediate in pyruvate kinase. The enzymatic reaction pro-
duces enol-pyruvate, which is in equilibrium with pyruvate. As indicated by the relative lengths
of the equilibrium arrows, pyruvate is the predominant form.
FIGURE 9.23 Covalent regulation of pyruvate kinase. Pyruvate kinase is subjected to a

phosphorylation reaction, catalyzed by protein kinase A. The phosphorylated enzyme, in turn,
becomes less active. A protein phosphatase catalyzes the hydrolysis of the phosphoenzyme,
with release of Pi.
already considered. One feature of the reaction is that the direct enzymatic product is enol-
pyruvate, which forms an equilibrium with pyruvate (Figure 9.22). That equilibrium, known
as a keto-enol equilibrium, greatly favors pyruvate, but a small amount of the enol form is
present in all such ketones.
Pyruvate kinase is a metabolically irreversible reaction that is regulated both allosterically
and by covalent modification. The key allosteric regulator of pyruvate kinase is fructose-
1,6-bis-P2, the product of phosphofructokinase (PFK), the prime regulatory enzyme of gly-
colysis. Thus, when PFK is more active, the concentration of fructose–1,6–bis–P2 rises and
activates pyruvate kinase, known as feed-forward activation.
In some tissues, such as the liver, pyruvate kinase is a substrate for phosphorylation.
Hormonal regulation may lead to an activation of the enzyme protein kinase A, which phos-
phorylates and inactivates pyruvate kinase (Figure 9.23). A separate enzyme, a protein phos-
phatase, catalyzes dephosphorylation and returns pyruvate kinase to its active form.
Both the allosteric stimulation and the covalent inhibition alter the kinetic pro-
file of pyruvate kinase (Figure 9.24), which resembles the strictly allosteric regulation of
FIGURE 9.24 Modulation of pyruvate kinase activity. Two means of regulating the activity of
pyruvate kinase exist. Covalent modification by phosphorylation causes a decrease in enzyme
activity, evidenced by the shift of the curve to the right. Allosteric binding of fructose-1,6-P2. the
product of PFK, activates the enzyme, and shifts the curve to the left. Because PFK participates in
an earlier step in the pathway, its product is a feed-forward activator of pyruvate kinase.
phosphofructokinase (Figure 9.8). Pyruvate kinase is considered a secondary regulatory site

of glycolysis. As a result of feed-forward activation, it is responsive to the primary control by
phosphofructokinase.
There are three known isozymes of pyruvate kinase: L, M1 and M2, where L (liver) and
M (muscle) represent the tissues in which the enzyme forms were first discovered. M1 and
M2 are formed from the same gene, but the resulting mRNA is spliced differently (splice
variants, Chapter 16). Most cells contain a form of the M isoform. Isozyme M2 is found
in neonatal and cancer cells. Ironically, these cells have an elevated rate of glycolysis, even
though this pyruvate kinase has a lower maximal velocity. A further distinction is that the
M2 form is also subject to phosphorylation and dephosphorylation with activity results simi-
lar to those of Figure 9.24. The residue subject to phosphorylation is a tyrosine rather than
a serine. Thus, the kinase involved is a phosphotyrosine kinase, and the phosphatase is a
phosphotyrosine phosphatase. Once suppressed by phosphorylation, the pyruvate kinase
substrate, PEP, is diverted into the serine biosynthesis pathway (see Chapter 15).
9.3 COMPLETING THE PATHWAY
If we consider the sequence of glucose to pyruvate as the sum of its connected parts, the
overall reaction is:

(9.5) Glucose + 2 ADP + 2 Pi + 2 NAD+ ® 2 pyruvate + 2 ATP + 2 NADH
Glucose and pyruvate appear at the ends of the pathway, while the other molecules in this
equation are mobile cofactors. The cofactors are present in limited concentrations and must
be continuously regenerated for the pathway to operate. By contrast, glucose is continually
supplied and pyruvate consumed by another pathway. We have a complete pathway only if
the mobile cofactors are balanced.
The cofactors of glycolysis can be grouped into two categories:
1. Phosphorylation cofactors: ATP, ADP, and P.

2. Redox cofactors: NAD+ and NADH.
Energy-utilizing reactions regenerate the phosphorylation cofactors. ATP is the central

energy intermediate of cells. Thus, a prime function of glycolysis is to provide that energy. For
200 9.3 Completing the Pathway
example, the Na+/K+-ATPase that catalyzes the export of cellular Na+ and import of K+ is one
reaction that turns over the phosphorylation cofactors of glycolysis. The Na+/K+-ATPase is
thus linked in parallel to glycolysis, as are all of the energy-utilizing steps of the cell cytosol.
The redox cofactor NADH must be reoxidized to NAD+ so that glycolysis can continu-
ously operate. Lactate formation is the predominant route, as is discussed next. We will then
consider an alternative to lactate: the conversion of pyruvate to ethanol.
9.3.1 LACTATE FORMATION
The most common end product of glycolysis is lactate, the product of the near-equilibrium
enzyme lactate dehydrogenase (lactate DH), shown in Figure 9.25. The formation of lactate
is a dead-end in glucose metabolism. It is not connected to any other reaction and exits the
cell for use in other cells.
There are several isozyme forms of lactate DH, so that different proteins are present, for
example, in the liver, muscle, and heart. A clinical test that takes advantage of this distinc-
tion involves the analysis of lactate DH in the blood and separation of the isozyme forms by
electrophoresis. An elevated heart lactate DH indicates the rupture of heart cells that caused
the release of the enzyme into the bloodstream. However, the isozyme differences have no
regulatory significance because the enzyme is near-equilibrium in all of these tissues and
thus has no influence on the extent to which pyruvate and lactate are interconverted.
9.3.2 ETHANOL FORMATION
Some organisms, such as yeast, can convert glucose to ethanol instead of lactate. This process
is also a form of glycolysis, but it is more commonly called fermentation. The pathway lead-
ing from glucose to pyruvate in yeast is the same as we have documented above.
Pyruvate is converted to ethanol in two steps. First, pyruvate is decarboxylated in a reac-
tion catalyzed by pyruvate decarboxylase:
(9.6) Pyruvate ® Acetaldehyde + CO2
This step is metabolically irreversible, as are most reactions that release CO2. Irreversibility
results from the fact that CO2 is a gas, and its release from the solution phase pulls the reac-
tion forward. Pyruvate decarboxylase contains the bound cofactor thiamine pyrophosphate;
the mechanism of pyruvate decarboxylase is outlined in Figure 7.15.
The second step in the conversion of pyruvate to ethanol in yeast is catalyzed by
alcohol DH:
(9.7) Acetaldehyde + NADH Ethanol + NAD+
which provides NAD+ for glyceraldehyde phosphate DH and allows the pathway to continue
another cycle. The essence of the hydride transfer to NAD+ was illustrated in Figure 7.2. The
mechanism of alcohol DH is shown in Figure 9.26. Alcohol DH is the second example of a
metalloenzyme; the first was enolase. In the alcohol DH mechanism, a Zn2+ ion assists in
FIGURE 9.25 The lactate DH reaction

FIGURE 9.26 Mechanism of alcohol DH. Alcohol DH has a bound Zn2+ ion, which forms a
chelate bond to the substrate in the process of electron flow from NADH. The formation of the
chelate drives the electron flow and stabilizes the intermediate. A base from the enzyme adds a
proton to form the product, ethanol.
hydride transfer, forming a chelate bond to the oxygen in the intermediate. A proton transfer
from a separate basic group on the enzyme to form the alcohol group of the product com-
pletes the mechanism.
Besides yeast, alcohol DH is also present in the livers of humans (and many other ani-
mals) and is responsible for the oxidation of ethanol as well as other alcohols in metabolic
pathways. The mammalian liver isozyme has a high K M for ethanol, and typically acts in the
direction of acetaldehyde formation (i.e., in the reverse direction to yeast fermentation).
The products of yeast fermentation are thus CO2 and ethanol. The first product accounts
for the action of “baker’s yeast”, which causes bread dough to rise. The second is the basis
of the centuries-old alcoholic beverage industry. In the production of beer and champagne,
both fermentation products – CO2 and ethanol – remain. In wine, the CO2 must be drawn
off during the fermentation process. Yeast is unable to survive alcohol concentrations much
above 10%; hence, chemical distillation is needed to produce high-alcohol content beverages
such as whiskey and gin.
9.4 ENERGETICS OF GLYCOLYSIS
Balancing the phosphorylation cofactors of glycolysis requires energy-utilizing pathways, as

we have observed. Thus, one function of glycolysis is to provide energy as ATP for other
pathways. The net yield of energy from glycolysis is 2 ATP to convert 1 glucose to 2 lactate
molecules.
In this section, we first examine the thermodynamics of the overall pathway of glycolysis,
extending our study of individual reactions in Chapter 7. Next, we consider three situations
that affect the energetics of glycolysis: a shunt pathway that exists in red blood cells, a form
of arsenic poisoning, and the metabolism of fructose by the liver.
9.4.1 PATHWAY THERMODYNAMICS
Values for the equilibrium constants of the individual glycolytic reactions are well known,
and the concentrations of intermediates have been established in many cell types. The red
blood cell is unique because glycolysis is the only means of ATP production; there are few
other pathways, and no intracellular vesicles. Thus, the analysis of glycolysis in this cell type
is relatively uncomplicated.
Figure 9.27 presents pathway data for both the standard free energy changes (ΔGo) of the
glycolytic reactions as well as the ΔG values measured from concentrations of compounds
202 9.4 Energetics of Glycolysis
FIGURE 9.27 Thermodynamics of glycolysis. The upper graph is a plot of standard free ener-
gies, and the slopes between the points representing glycolytic intermediates indicate the
changes in free energy visually for each step. The ΔGo values are positive in some cases. Thus,
under standard conditions, the pathway cannot go forward. The bottom graph is a plot of actual
free energies. In this case, all of the slopes are negative. Three steps – hexokinase, phosphofruc-
tokinase, and pyruvate kinase – have strongly negative ΔG values, corresponding to the three
metabolically irreversible reactions of glycolysis.
in red blood cells undergoing glycolysis. The diagram is a visual display of reaction energies
as well as overall pathway changes. The ordinate of the plot is “G” and “Go”, and the abscissa
consists of the glycolytic pathway compounds in sequence.
Because only changes in thermodynamic variables are available, rather than absolute val-
ues, the points are plotted by arbitrarily setting the Go value of glucose to zero. The G value
for glucose is set lower than the Go value, reflecting a change in concentration from 1 M to
millimolar concentrations.
With the first points established, the other data points are derived from the free energy
changes for the reaction between the pathway intermediates. For example, the ΔGo value
for hexokinase represents the value recorded as the point corresponding to G6P in the
figure. A similar procedure was applied to the lower line, using measured ΔG values. The
line connecting any two points thus represents the value of the free energy change, in
both direction (a positive slope shows a positive change; a negative slope shows a negative
change) and quantity (a steep slope shows a large change; a shallow slope shows a small
change).
The changes in ΔGo can be judged visually by comparing the slopes for the glycolytic
reactions. Because ΔGo values can be negative or positive as we move from glucose to lactate,
this pathway is not thermodynamically possible under standard conditions. Nevertheless,
the overall change in ΔGo is negative, so the overall change is allowed thermodynamically
under standard conditions, but not by the glycolytic route. Despite the fact that the overall
standard free energy is negative, glycolysis cannot proceed under standard conditions. As
indicated in the lower trace, each step has a negative ΔG, which means this pathway is ther-
modynamically allowed. No step has a positive or a zero ΔG value. If it were positive, the
reverse reaction would occur. If it were zero, the reaction would be at equilibrium, and no net
reaction would occur. It cannot be argued that an ensuing reaction could somehow pull an
unfavorable one to completion. Instead, the prevailing concentrations in the cell determine
the status of the free energy, as we have already observed from the free energy equation of
Chapter 8.
A further important piece of information can be gleaned from Figure 9.27: most of the
slopes of the actual free energy plot are relatively shallow, visually indicating the near-
equilibrium reactions. Only three are steep: hexokinase, phosphofructokinase, and pyruvate
kinase. These correspond to the metabolically irreversible steps of the pathway.
9.4.2 RED BLOOD CELL SHUNT PATHWAY
Recall that 2,3-bis-P-glycerate decreases the affinity of oxygen for hemoglobin at high alti-
tudes (Chapter 5). The formation of this compound is an example of a shunt pathway, because
it originates from one glycolytic intermediate and terminates in another.
The pathway begins with the glycolytic intermediate 1,3-bis-P-glycerate, the product of
the glyceraldehyde-P dehydrogenase step. Red blood cells have a mutase enzyme that cata-
lyzes the reaction:
O O
C OP C O
(9.8) H C OH H C OP
CH2OP CH2OP
1,3-bis-P-glycerate → 2,3-bis-P-glycerate
The mechanism of this mutase is essentially identical to that of the P-glyceromutase reac-
tion (Figure 9.17), which shuttles phosphoryl groups between the substrate and a histidyl
group on the enzyme. A phosphatase reaction catalyzes the destruction of this regulator:

(9.9) 2, 3-bis-P-glycerate ® 3-P-glycerate + Pi
This phosphatase activity is catalyzed by the same protein that has mutase activity. Hence,
the concentration of the regulator in the cell depends on a balance between two distinct
activities of the same enzymatic protein, a mutase/phosphatase. This situation is similar to
the protein phosphofructokinase–2/fructose-2,6-P2, which catalyzes both the formation and
the destruction of fructose-2,6-P2.
There is an energy consequence to flow through the red blood cell shunt pathway because it
bypasses the P-glycerate kinase reaction (Figure 9.28). Because a complete bypass of this reac-
tion would result in zero ATP production, and as glycolysis is the only means of energy for-
mation for red blood cells, flow through the mutase/phosphatase must be kept relatively low.
9.4.3 ARSENATE POISONING
Elemental arsenic is positioned just below phosphorus in the same column (group 5A)
of the periodic table. This means that the outer electron configuration of As and P is the
same, and we would expect the elements to have some chemical properties in common.
These include the ability to form similar oxides. By analogy to phosphate (PO42−), arsenate
(AsO42−) can substitute in the glyceraldehyde-P DH reaction in place of phosphate, producing
1-arseno-3-P-glycerate:
O O
C OPO32- C OAsO32-
H C OH H C OH
CH2OP CH2OP
1,3-bis-P-glycerate 1-arseno-3-P-glycerate
FIGURE 9.28 The red blood cell bypass. The multifunctional mutase/phosphatase enzyme
found in red cells forms and removes 2,3-bis-P-glycerate. The latter causes a decreased affinity of
hemoglobin for oxygen, appropriate for high altitudes. Notice that flux through the shunt also
bypasses the formation of ATP. To the extent that the shunt is operating, it decreases the yield of
ATP from glycolysis.
FIGURE 9.29 Arsenolysis of 1-arsenate-3-P-glycerate. The nonenzymatic hydrolysis produces

the same glycolytic intermediate – 3PG – as the normal glyceraldehyde P DH reaction.
The arsenate ester is rapidly and nonenzymatically hydrolyzed to 3-P-glycerate

(Figure 9.29). The reactions with arsenate therefore represent a bypass between GAP and
3PG as illustrated in Figure 9.30. Both the regular route of glycolysis and the bypass in the
presence of arsenate convert GAP to 3PG. However, the arsenate route does not provide
ATP formation. Arsenate is therefore not an inhibitor of glycolysis, but rather an uncou-
pler of the pathway. An uncoupler allows pathway flow but lowers the amount of ATP for-
mation. If all of the carbon through glycolysis flowed through the arsenate pathway, zero
net ATP formation would result. Uncoupling is a situation more commonly encountered
in studies of mitochondrial energy production. While there are multiple explanations for
arsenic toxicity, most proposed actions share the ability of arsenate to mimic phosphate
in cells.
FIGURE 9.30 Modified glycolysis in the presence of arsenate. The presence of arsenate pro-
vides an alternative pathway, also using the glyceraldehyde-P DH reaction. However, this also
bypasses ATP formation.
9.4.4 FRUCTOSE METABOLISM
Fructose is a common dietary sugar. Until the 1980s, most dietary sugar was sucrose, a disac-
charide consisting of glucose and fructose. In the last few decades, most sucrose has been
replaced by high-fructose corn syrup, a mixture of free glucose and fructose (Box 9.4). A
small amount of fructose is metabolized by many cells because some GLUT isoforms trans-
port it and because it is a poor substrate of some hexokinase isozymes. However, most fruc-
tose is metabolized through a different pathway that is unique to the liver.
In the liver, fructose is transported across the plasma membrane via GLUT2. Intracellular
fructose is converted to fructose-1-P, catalyzed by the enzyme fructokinase (Figure 9.31).
Box 9.4 High-Fructose Corn Syrup
Most of our dietary sugar is derived from corn rather than the sucrose that is extracted
from sugar cane and sugar beets. An industrial process enzymatically hydrolyzes corn-
starch to glucose, followed by enzymatic isomerization of some of the glucose to fruc-
tose. The resulting solution, abbreviated HFCS, is cheaper to produce than sucrose due
to the abundance of corn. HFCS is thus merely a mixture of glucose and fructose, and can
be metabolized even more rapidly than sucrose because it obviates the need for sucrose
hydrolyze to glucose and fructose. There are some indications that HFCS is nutritionally
distinct from sucrose and that it may be more efficiently converted to fat. However, it is
difficult to separate nutritional from sociological factors. For example, the cheaper “sugar
substitute” is present in ever larger (“supersized”) portions of beverages and fast food. It
seems likely that both metabolic and behavioral factors contribute to obesity.
FIGURE 9.31 Metabolism of fructose in the liver. Three special liver enzymes allow fructose
carbon to enter glycolysis. First, fructokinase catalyzes the conversion of fructose to fructose-1-P.
Second, aldolase B catalyzes the splitting of fructose-1-P into dihydroxyacetone-P and glyceral-
dehyde. Third, glyceraldehyde is a substrate for triose kinase, which catalyzes the formation of
glyceraldehyde-P. While fructose metabolism has the same balance of ATP as glucose, the major
regulatory step of glycolysis, PFK, is bypassed. This escape from pathway regulation accounts for
the rapid metabolism of fructose.
Next, aldolase B, a liver isozyme, catalyzes the aldol cleavage of fructose-1-P to dihydroxy-
acetone-P and glyceraldehyde (analogous to the aldolase reaction of glycolysis, Figure 9.11).
Glyceraldehyde is a substrate for the triose kinase reaction, catalyzed by a third unique liver
enzyme, which converts glyceraldehyde to glyceraldehyde-3-P. From this point forward,
the carbon of fructose is metabolized using the usual glycolytic steps. Note that the ATP
balance – that is, the difference between ATP input and ATP production – matches that of
glucose, despite using different steps (i.e., triose kinase in place of P-fructokinase).
A significant distinction between fructose metabolism and glucose metabolism is that
fructose metabolism bypasses the PFK reaction, which is the rate-limiting step for glucose
metabolism. The connections between fructose metabolism and glycolysis are diagrammed
in Figure 9.32. Due to the absence of control by PFK, the entry of fructose into glycolysis is
more rapid and less responsive to regulation compared to glucose as substrate. As a further
consequence, carbon from fructose can enter more rapidly into fat synthesis, contributing
to obesity. A separate complication results from the ingestion of a large bolus of fructose.
Phosphorylation to fructose-1-P can be so rapid that the cell’s ATP supply is depleted to the
point where the cell does not survive.
9.4.5 ENERGY BALANCE AND GLYCOLYTIC CONNECTIONS
We have now considered three situations that affect ATP production by glycolysis: 2,3-bis-
phosphoglycerate formation in the red cell shunt, arsenic poisoning, and fructose metabolism.
FIGURE 9.32 Fructose catabolism in liver. Fructose is ultimately converted to DHAP and glyc-
eraldehyde; both intermediates enter glycolysis at the triose level.
The first is a controlled metabolic event that occurs during adjustment to lowered oxygen
concentrations, such as at high altitudes. As a result, flux through glycolysis is ensured as the
cell controls the amount of regulator produced. The second is a poisoning situation, which
depends only on the amount of arsenate present. Arsenate can increase the flux of glycoly-
sis while reducing the rate of ATP formation. Finally, the rapid phosphorylation of fructose
can deplete cellular ATP levels and lead to cell death. Thus, each has distinct alterations of
energy balance. In contrast, we found the formation of the PFK regulator by the bifunctional
enzyme PFK-2 is also a bypass of glycolysis, but this occurs at the hexose level, and does
not consume significant quantities of ATP. Most importantly, fructose 2,6-P formation is of
much lower magnitude than FBP formation, so that its effect on glycolysis is minimal.
9.5 METABOLIC CONNECTIONS TO GLYCOLYSIS
Glycolysis is a central pathway in virtually all cells and has extensive connections to other
pathways. In our final section of glycolysis, we consider separate ways of entering glycolysis,
connections to intermediates, and multiple pathway end products.
9.5.1 ALTERNATIVE ENTRY POINTS
We have just examined fructose metabolism from the standpoint of its effects on energetics.
At the same time, we can think of fructose as a distinct entry point to glycolysis. Another
common sugar and a possible starting point for glycolysis is galactose, derived from the milk
sugar, lactose. Glycolysis can also originate from the polymer glycogen (Chapter 13).
9.5.2 GLYCOLYTIC INTERMEDIATES AS INTERSECTION POINTS
In keeping with its ubiquitous occurrence in cells and its ancient origins, the intermediates of
glycolysis are commonly used as intermediates of other pathways. For example, the red blood
208 Summary
cell 2,3-P2-glycerate shunt described in the previous section could be viewed as a pathway
that begins with one intermediate of the pathway and ends with another. Another example
is the pentose phosphate shunt (Chapter 13). The formation of the regulator fructose-2,6-P2
can be considered as a pathway that begins and ends with the same glycolytic intermediate.
The utilization of glycolytic intermediates in other pathways is more extensive than this one.
In fact, all of the intermediates of glycolysis are used in other pathways, many of which are
examined elsewhere in this text.
9.5.3 ALTERNATIVE ENDPOINTS OF GLYCOLYSIS
We have seen that one alternative endpoint of glycolysis is ethanol formation, limited to yeast
and a few other microorganisms. A significant glycolytic endpoint in mammals is the con-
version of pyruvate to alanine. Virtually all cells have the pathway for the complete oxidation
of pyruvate, which we take up in the following chapter.
SUMMARY
Glycolysis is the best-known pathway in cells, and serves as a model for understanding meta-
bolic features, such as enzyme chemistry, mobile versus bound cofactors, energy links to
pathways, and regulatory steps. The details of enzyme mechanisms show many of the fea-
tures repeated throughout metabolism, such as nucleophilic substitution, acid-base catalysis,
and electron rearrangement in isomerase reactions. The two mobile cofactor pairs in gly-
colysis are ATP/ADP and NAD+/NADH. The first connects glycolysis to energy production
because net ATP is produced by the pathway and is used in other cellular reactions, such as
maintaining the plasma membrane sodium gradient. The second is the redox cofactor pair,
which is balanced if glycolysis converts glucose to lactate. The most important regulatory
step of glycolysis is the reaction catalyzed by phosphofructokinase, which is stimulated by
fructose-2,6-P2 (reflecting hormonal control) and inhibited by citrate (reflecting feedback
regulation from oxidative metabolism). A secondary regulatory point, pyruvate kinase, is in
part controlled by feed-forward activation by fructose-1,6-P2, the product of phosphofruc-
tokinase. Because all of its intermediates intersect with other pathways, glycolysis can be
thought of as the crossroads of metabolism.
REVIEW QUESTIONS
1. Explain why fructose has the same ATP balance as glucose.

2. Pancreatic cells secrete insulin in response to glucose. Here GLUT2 plays an impor-
tant role as part of the “glucose sensor”. Explain.
3. When kinase reactions are measured in vitro, all of them are inhibited by ATP.
However, the concentration of ATP in cells is virtually constant. Can ATP be consid-
ered a regulator of glycolysis?
4. What is the effect of arsenate ingestion at high altitudes, where the red cell shunt
mutase is more active?
5. In the liver, pyruvate kinase is a substrate of protein kinase A, but so is PFK-2. Explain
how both of these will affect the rate of glycolysis.
6. Explain why arsenate can increase the rate of glycolysis, and yet decrease ATP forma-
tion. Would arsenate also have this effect if fructose was the substrate?
7. The P-glycerate kinase reaction is near-equilibrium, yet it leads to the production of
ATP, a high-energy intermediate. Explain this conundrum.
8. Both NADH and TPP are enzyme cofactors; however, TPP does not rely on other
reactions for its regeneration. Why is this so?
9. The product of the enolase reaction would appear from the reaction mechanism to be
an enol rather than pyruvate. How can this be explained?
FIGURE CA9.1 Reactions of the Entner–Doudoroff pathway. G6P is formed through a hexo-
kinase reaction. Subsequently, oxidation at C1 is catalyzed by a dehydrogenase that uses NADP+
as the mobile redox cofactor. The splitting of the substrate of this reaction, 6-P-gluconate, is
catalyzed by an aldolase. The remainder of the reactions between GAP and pyruvate are the
same as those of glycolysis.
CHAPTER 9 ADDENDUM: ALTERNATIVES TO GLYCOLYSIS
Variations of the glycolytic route have been presented in this chapter as bypasses or entry
reactions to demonstrate connections in multicellular organisms. As pointed out, yeast path-
way alters only the final reaction: pyruvate is converted to ethanol rather than lactate. A
different variation to the pathway that also produces ATP anaerobically is widely present in
bacteria and recently found in plants: the Entner–Doudoroff pathway (ED). The distinctive
reactions are illustrated in Figure CA9.1.
The formation of G6P is accomplished by a hexokinase, as in the glycolytic pathway. The
next reaction is catalyzed by glucose-6-P DH, which is also found in photosynthetic organ-
isms as part of the carbon reactions (Chapter 12) and higher organisms as part of the pen-
tose phosphate shunt (Chapter 13). The subsequent cleavage to form pyruvate and GAP
is catalyzed by an aldolase reaction, with a similar mechanism to the aldolase of glycolysis.
Reactions between GAP and pyruvate are the same as for glycolysis.
The pathway produces both NADPH as well as NADH (the latter at the glyceraldehyde-P
DH reaction as in the usual glycolytic pathway); these must be recycled in other reactions
to ensure continued traversal of the Entner–Doudoroff pathway. It should also be noted that
ATP production is less than glycolysis: 1 ATP is consumed at hexokinase, and 2 ATP are
formed between GAP and pyruvate, for a net gain of 1 ATP. On the other hand, the genera-
tion of NADPH can be utilized in synthetic reactions, as described later in this text.
KEY TERMS
carbon reactions
complete pathway
electron sink
feed-forward activation
fermentation
glucokinase
isozymes
near-equilibrium
Pasteur effect
pentose phosphate shunt
splice variants
210 Bibliography
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The Krebs Cycle 10
Thirty years after discovering glycolysis, investigators had identified several intermediates
involved in the complete oxidation of pyruvate to CO2. However, the route itself remained
a mystery until Hans Krebs found that pyruvate entered into a reaction with a four-car-
bon intermediate, producing citric acid, a six-carbon tricarboxylic acid. He also showed
that after a series of reactions, the same four-carbon intermediate was regenerated; in other
words, the pathway was a cycle. The Krebs cycle, now considered a landmark study, was
published in 1937 as the citric acid cycle (Box 10.1). The result was not well-received, how-
ever, for reasons that we explore in this chapter. In fact, many called the route the “Krebs
cycle” not as praise but to suggest that it was just one investigator’s belief. The pathway’s
cyclic nature was tentatively accepted, but only the part involving tricarboxylic acid inter-
mediates other than citrate. The pathway was thus referred to as the tricarboxylic acid cycle.
Today, the route is well established (Figure 10.1), and all three names are used. We choose the
Krebs cycle, as it is pithier, vindicates the findings of a great biochemist, and conforms to the
tradition of other named highlights of biochemistry, such as Michaelis–Menten kinetics, the
Lineweaver–Burke plot, and the Calvin cycle.
10.1 A CYCLIC PATHWAY
A series of enzymatic reactions in a cyclic pathway is analogous to the individual steps in an

enzymatic reaction. Consider two examples of enzyme reaction mechanisms involved in the
formation of ethanol from pyruvate by yeast, drawn in Figure 10.2 as cyclic processes. The
first reaction (Figure 10.2a) shows pyruvate decarboxylase (E1) and its other enzyme forms,
E1:pyruvate and E1:acetaldehyde, as cycle intermediates. Pyruvate enters the cycle, and the
products, CO2 and acetaldehyde, leave. For the second reaction (Figure 10.2b), the cycle inter-
mediates are alcohol dehydrogenase (E2), E2:acetaldehyde and E2:ethanol. Acetaldehyde and
NAD+ enter the cycle, and NADH and ethanol leave.
Every enzymatic reaction can be written as a cycle, which means that – theoretically at
least – just one molecule of the enzyme can catalyze infinitely many conversions of a sub-
strate to product. In reality, enzymes must accumulate some concentration of substrate to
achieve appreciable activity, and enzymes have a finite lifespan. Still, relative to the flow of
substrate to product, tiny amounts of the enzyme are needed. Alternatively, we could say that
the enzyme is required in catalytic amounts. Every catalytic process can be drawn as a cycle.
Figure 10.2c is a diagram of a metabolic cycle. Inputs and exits from the cycle are simi-
lar to the enzymatic cycles. However, the intermediates are not enzyme forms, but instead
pathway intermediate molecules. Each step within the metabolic cycle is itself catalyzed by
an enzyme. Like an enzymatic cycle, the amount of intermediates required for the operation
of the cycle is minuscule compared to the flow of substrate to product, here illustrated as
acetyl-CoA to CO2. The role of mobile cofactors, shown for NAD+ and NADH, is the same
as for any other pathway. While both the substrate (acetyl CoA) and the cyclic intermediates
are both pathway intermediates, they have very different behavior. Only acetyl-CoA is con-
verted to products; all pathway intermediates are regenerated. The pathway intermediates
are present in catalytic amounts. Figure 10.2c is an abbreviated form of the Krebs cycle but
does illustrate the critical point that acetyl-CoA is the sole substrate of the Krebs cycle, and
CO2 is the pathway product.
211
212 10.2 Acetyl-CoA: Substrate of the Krebs Cycle
Box 10.1 Word Origins: Krebs Cycle Intermediates
Many names of Krebs cycle intermediates come from the organism in which the com-
pound was first discovered. Citrate is the most transparent, derived from citrus fruits.
Aconitate is a compound from aconite (monkshood), a member of the buttercup fam-
ily. Succinate was first found in amber, a fossilized resin (the Latin for this is succinum).
Fumarate is found in plants of the fumaria genus (poppy family). Malate is an acid found in
abundance in the Malus genus (crabapple). While all of these organisms do have a Krebs
cycle, the abundance of the compounds is due to specialized pathways (called second-
ary metabolism), which protect plants from predators. The naming of other compounds
is more prosaic, as they were already known to scientists at the time of their recognition
as Krebs cycle intermediates, α–ketoglutarate and oxaloacetate.
FIGURE 10.1 Reactions of the Krebs Cycle. The cycle intermediates and their structures are
shown as a cycle; acetyl-CoA is the substrate. Cofactors are shown in blue.
10.2 ACETYL-CoA: SUBSTRATE OF THE KREBS CYCLE
The overall operation of the Krebs cycle oxidizes the two acetyl carbons of acetyl-CoA com-
pletely to CO2, and high-energy electrons are captured in the cofactors nicotinamide adenine
dinucleotide (NADH) and ubiquinone (UQ). These cofactors then become substrates for the
next pathway in metabolism, the electron transport chain.
The molecule acetyl-CoA is a thioester of acetate with CoA. Most significantly, it has a
free –SH group at the end of the molecule, which can engage in readily reversible thioester
formation. Often, the cofactor is written to emphasize the sulfur moiety, as CoA-SH.
The formation of acetyl-CoA from pyruvate requires an enzyme complex exclusively
found in the mitochondria. However, pyruvate is formed through glycolytic reactions that
occur in the cytosol.
Chapter 10 – The Krebs Cycle 213
FIGURE 10.2 Enzymatic and Metabolic Cycles. Every catalytic event can be written as a cycle.
The enzymes (a) pyruvate decarboxylase and (b) alcohol DH, the final steps of yeast fermenta-
tion, have the substrates pyruvate and acetaldehyde as inputs and include CO2 and NADH as
outputs. An abbreviated Krebs cycle (c) shows a similar situation, except that the intermediates
of the Krebs cycle are pathway intermediates rather than enzyme-bound compounds. Like enzy-
matic cycles, the pathway represents a catalytic event, with a substrate as input, and CO2 and
reducing equivalents as output.
Cytosolic pyruvate must cross both the outer and the inner mitochondrial membrane
(Figure 10.3). Pyruvate crosses the outer membrane through the channel protein VDAC, or
voltage-dependent anion channel (Box 10.2). Despite its name, the channel is freely perme-
able to small molecules with a molecular weight up to about 10,000. On the other hand, the
inner membrane is impermeable to most molecules, so pyruvate entry requires a specific
transport protein. Known as the pyruvate transporter, the protein co-transports pyruvate
(charged −1) with a proton into the matrix space, the most interior portion of the mitochon-
dria. In the matrix space, subsequent oxidation takes place.
The first step in this oxidation is the conversion of pyruvate to acetyl-CoA, catalyzed by
the pyruvate dehydrogenase complex. An enzyme complex is a group of enzymes that
catalyzes a metabolic sequence without releasing intermediates. The enzymes are closely
associated with one another, and they act as if a single reaction was taking place. The overall
reaction catalyzed by the pyruvate dehydrogenase complex is:

(10.1) Pyruvate + CoA + NAD+ ® Acetyl-CoA + NADH
214 10.2 Acetyl-CoA: Substrate of the Krebs Cycle
Mitochondirion
Outer membrane
Inner membrane
Matrix
Intermembrane
space
Matrix
Inner –
membrane H+
Intermembrane
space
–
Outer H+
membrane
Pyruvate VDAC
–
Outside of mitochondirion
FIGURE 10.3 Pyruvate Transport by Mitochondria. The outer membrane of mitochondria is

not a barrier for small molecules (i.e., those less than 10,000 Daltons) due to the presence of
porin channels. Hence, pyruvate readily crosses into the inter-membrane space. Pyruvate is trans-
ported along with H+ by a protein embedded in the inner membrane. Because pyruvate has a -1
charge, no change in electrical charge of the matrix occurs as a result of this transport.
Box 10.2 VDAC, Porin, and Outer Membrane Permeability
Multiple copies of the protein VDAC line the outer membrane of mitochondria and pro-
vide an entry pathway for small molecules, but not proteins, into the inter-membrane
space. A closely related protein, porin, serves the same function in bacteria. As mito-
chondria are evolutionarily derived from bacteria, the mitochondrial protein is some-
times called porin as well. The name VDAC suggests a functional role for selectivity (anion
channel) as well as control (voltage-dependent). However, neither of these functions has
been established; the voltage dependence is measured in protein subunits incorporated
into artificial lipid layers. While some cytosolic proteins bind VDAC in vitro¸ there is no
evidence of its metabolic significance. There is strong evidence showing that alterations
in VDAC lead to programmed cell death, or apoptosis, as the opening of the channels to
release cytochrome c is a critical step in this process. From the standpoint of pyruvate
entry into the mitochondria, the outer membrane is permeable without restrictions in a
way distinct from a near-equilibrium exchange: any molecule can pass through the chan-
nel without regard to structure, up to a molecular weight of 10,000.
This metabolically irreversible conversion involves three separate enzymes and several cofac-
tors. The mobile cofactors CoA, NAD+, and NADH appear in Equation (10.1). The others are
bound cofactors (prosthetic groups) that stay attached to enzymes of the complex over the
entire catalytic cycle: thiamine pyrophosphate, lipoic acid, and flavin adenine dinucleotide
(FAD; and its reduced form, FADH2 (see Chapter 7)). The bound cofactors do not appear in
the overall reaction; they remain attached to the individual enzymes of the complex. We have
already encountered thiamine pyrophosphate in the discussion of pyruvate decarboxylase. It
plays a nearly identical role in the pyruvate dehydrogenase complex.
The three enzymes catalyzing the conversion of pyruvate to acetyl-CoA are represented
as E1, E2, and E3 (detailed in Table 10.1). Note the presence of two other enzymes involved in
regulating the complex, discussed in Section 10.6.
The reaction mechanism is outlined in Figure 10.4. E1 first reacts with pyruvate through
the same mechanism as yeast pyruvate decarboxylase, except that the two-carbon fragment
is not released. A “swinging arm” of the complex – the lipoamide attached to E2 – removes
the two-carbon fragment, forming an acetyl-thioester and regenerating the free form of E1.
Next, the mobile cofactor CoA-SH displaces the acetyl group from E2:lipoamide, producing
the product, acetyl-CoA. After transferring the two-carbon fragment, the lipoamide is left in
a reduced (sulfhydryl) form. The remainder of the reactions of the complex serves to regener-
ate the enzyme forms. The sulfhydryls of E2:lipoamide reacts with the FAD of E3, producing
the oxidized lipoamide (the original form of E2) and reduced E3, bound to FADH 2. Finally,
the mobile cofactor NAD+ reacts with E3, returning it to a bound FAD form and releasing
NADH.
TABLE 10.1 Enzymes of the Pyruvate Dehydrogenase Complex

Name Alternative Name or Description Bound Cofactor Mobile Cofactor
E1 Pyruvate dehydrogenase TPP −
E2 Acetyl transferase Lipoamide –
E3 Lipoamide DH FAD NAD+, NADH
PDH Kinase Kinase acting on E1 – ATP, ADP
PDH Phosphatase Phosphatase acting on E1 – Pi
FIGURE 10.4 "Swinging Arm" Mechanism of the Pyruvate Dehydrogenase Complex. The first
portion of the mechanism, from E1 to its bound two-carbon fragment is the same as yeast pyru-
vate decarboxylase. However, rather than releasing this to solution, it is instead transferred to a
thioester of the lipoic acid of E2. Next, the "arm" of E2-which is lipoic acid in an amide link to a
lysine residue-reacts with CoA, releases the product acetyl-CoA, and leaves the enzyme with its
lipoic acid bound in the reduced, disulfide form. The sulfhydryls are reoxidized back to the disul-
fide form through the E3-bound FAD, thus producing E3:FADH2. E3:FADH2 is then reoxidized by
transferring a hydride to NAD+, releasing NADH, and reforming the oxidized E3:FAD. Thus, every
enzyme is returned to its original form so that another round of catalysis may occur.
216 10.3 Overview of Carbon Flow
10.3 OVERVIEW OF CARBON FLOW
Figure 10.5 shows the intermediates of the Krebs cycle as six-, five-, and four-carbon com-
pounds. As input to the pathway, the two-carbon segment of acetyl-CoA (indicated as con-
nected dark circles) condenses with a four-carbon compound to form a branched six-carbon
intermediate. The first carbon released as CO2 comes from the four-carbon (C4) fragment,
not the incoming acetyl-CoA. Subsequently, a second CO2 is released, which is also not
derived from the incoming acetyl-CoA. The resulting C4 intermediate is converted to a mol-
ecule with a plane of symmetry as indicated.The top and bottom halves of the molecule are
no longer distinct, indicated by the partial shading. In the second turn of the cycle, the two
carbons released represent some of the carbon that entered in the previous turn. This pattern
continues; ultimately, all of the incoming acetyl-CoA carbon is released as CO2. This feature
has confounded many radiotracer studies of the carbons of the Krebs cycle.
The curious pattern of carbon flow out of the Krebs cycle is a pattern that is also found
in other pathways, such as the catabolism of the sugar galactose discussed in Chapter 13.
The net flow can be understood if we focus on the steady-state situation instead of a sin-
gle turn through the cycle. Overall, the Krebs cycle will completely oxidize the two car-
bons of acetyl-CoA to CO2, but this will require many turns. We will refer to this feature as
repetition–variation, a term borrowed from the advertising industry. Ads must be repeated
to be effective, but to avoid monotony on the part of the buyer and the expense of producing
entirely new commercials, small portions are altered to create different versions. In the bio-
chemical case, repetition–variation provides an elegant means of ultimately fully oxidizing
the acetyl-CoA while also connecting intermediates to other metabolic pathways.
Having completed an overview of carbon flow, we turn next to a detailed examination of
the Krebs cycle reactions. We will examine the chemical conversions involved and the for-
mation of mobile cofactors, which represent energy extraction.
FIGURE 10.5 Carbon Fates in the Krebs Cycle. Acetyl-CoA is represented by the two filled
circles that are the substrate of the Krebs cycle. The first reaction is with the four-carbon, open-
circled substrate, to produce citrate, with the labeling pattern shown. Loss of both CO2 mole-
cules, as indicated, comes not from the input acetyl-CoA but rather from the carbon of the cycle
intermediate. Conversion to a symmetrical four-carbon intermediate prior to the next round of
synthesis means that half of the carbons released as CO2 in the next round originated from ace-
tyl-CoA in the previous round.
10.4 STEPS OF THE PATHWAY
With a cyclic pathway, it is reasonable to ask: where do we start? Since the input to the path-
way is acetyl-CoA, we can take the reaction it participates in as the first step.
10.4.1 CITRATE SYNTHASE
The citrate synthase reaction introduces new carbon into the pathway that is ultimately
released as CO2:
(10.2) Oxaloacetate + Acetyl-CoA ® Citrate + CoA
While the enzyme is metabolically irreversible, no allosteric modulators are known.

In the mechanism shown in Figure 10.6, extraction of a proton from the terminal methyl
group of acetyl-CoA allows electrons from the methyl end of the molecule to attack the car-
bonyl of oxaloacetate. This nucleophilic substitution leads to the citryl-CoA intermediate,
which is subsequently hydrolyzed to citrate.
FIGURE 10.6 Mechanism of Citrate Synthase. (a) Condensation of acetylCoA with oxaloac-
etate is initiated by base abstraction of a methyl proton and attack on the oxaloacetate carbonyl
by the resulting carbanion. The intermediate product is the CoA ester of citrate, which is hydro-
lyzed to the product, citrate. (b) Resonance forms of the carbanion of acetyl-CoA.
218 10.4 Steps of the Pathway
Methyl hydrogens are very stable, and a proton cannot usually be removed by a basic
group, shown in Figure 10.6a. However, the methyl carbon in acetyl-CoA is adjacent to a
carbonyl. As shown in Figure 10.6b, the carbanion is resonance-stabilized. Essentially, the
mechanism of citrate synthase involves the development of a resonance-stabilized carbanion
that undergoes an addition reaction with oxaloacetate.
10.4.2 ACONITASE
Aconitase catalyzes two sequential reactions: dehydration to cis-aconitate, followed by

hydration to isocitrate (Figure 10.7). The enzyme catalyzes the reactions at near-equilibrium,
and the chemistry itself is straightforward acid-base catalysis. Two features compel us to
examine the enzymatic steps in detail. The first is a new aspect of stereochemistry. Of the
three molecules displayed in Figure 10.7, isocitrate has two chiral centers, and the others
have none. The second feature is that the enzyme catalyzes two sequential reactions using
the same active site.
A bound cofactor in aconitase is the Fe–S Cluster, illustrated in Figure 10.8. The Fe2+
ions in this structure are liganded to sulfur atoms or a cysteine residue of the protein. In
aconitase, one of the Fe2+ ions forms a coordinate bond to one of the carboxyl groups of the
substrate, and also to a water molecule involved in catalysis. Fe–S clusters in other proteins
are redox-active, such as those involved in mitochondrial electron transport (Chapter 11).
FIGURE 10.7 Aconitase Reactions. Aconitase catalyzes two sequential reactions: the dehydra-
tion of citrate and the hydration of cis-aconitate.
FIGURE 10.8 Fe–S Cluster. One of the iron-sulfur clusters is shown. The bonds between S and
Fe2+ are coordinate covalent bonds, involving the d-orbitals of the Fe2+.
The mechanism of aconitase is outlined in Figure 10.9. On the left panel of the figure
are the straight-chain forms of the molecules involved: citrate, cis-aconitate, and isocitrate.
Three carbons are labeled, using the Greek lettering system for carbons based on their prox-
imity to a carboxyl group. Cis-aconitate is diagrammed twice, with one of the forms rotated
180°. The mechanism begins with the removal of a proton from the Cα position by an alk-
oxide from an enzymatic serine residue (Figure 10.9a). All of the hydrophilic groups of the
molecule – the three carboxylates and the hydroxyl group – are firmly attached to enzyme
residues (many of them arginyl groups); one of them is also coordinated to the Fe–S cluster.
The result of the acid-base reaction is the selective formation of the cis form of aconitate
(Figure 10.9b). An unusual feature of the mechanism is the flip, as indicated in producing the
structure of Figure 10.9c. This flip is necessary because the same active site – with the resi-
dues situated in the enzyme in the same places – is used to hydrate cis-aconitate, leaving the
hydroxyl group attached to the Cα. The electron rearrangement illustrated in Figure 10.9d
shows a water molecule chelated an Fe2+ of the Fe–S cluster. Removal of one of the protons
of water by the histidyl residue initiates attack of the hydroxyl group at Cα, and migration of
the π–electrons of the double bond to attach to the proton of the serine residue. The product
formation in Figure 10.9e leaves the enzyme in its original form.
The overall conversion of citrate to isocitrate was a contentious issue for a decade follow-
ing the original 1937 proposal by Krebs of a cyclic set of reactions that we now accept as the
major oxidative pathway of metabolism. The problem is one of stereochemistry. Despite the
acceptance of stereochemical isomers by that date, the notion that a chiral compound could
arise from an achiral compound – as the conversion of citrate to isocitrate would require –
was anathema to organic chemists.
COO
ser-E
α ser-E
H C H COO O
COO O H
β
HO C COO β
H OOC COO
β
OOC COO
γ γ
α
H C H γ α
H
O H
E-his H E-his (c)
COO H
citrate
(a)
COO COO
IP ser-E
α
C H
γ
FL COO
H C H O
β
H
OOC COO
βC COO Fe/S
β C COO
ser-E O α γ
γ
α COO H H H
H C H C H O
H (d)
β
E-his
COO OOC COO
COO
γ
cis-aconitate α
H
E-his ser-E
COO COO
(b) O
α
H C H OOC OH β
COO
β
α γ
H C COO
H H
γ
HO C H
E-his H (e)
COO
isocitrate
FIGURE 10.9 Aconitase Mechanism. Left panel: substrates and products of aconitase are shown, with carbons labeled relative
to one of the carboxylates of citrate. Cis-aconitate is duplicated as a “flipped” (180 degree rotated) molecule. Transformation of (a)
to (b) involves positioned ser and his residues of the enzyme, illustrated in blue. The “flip” to (c) is dissociation of cis-aconitate from
the enzyme and rebinding after a 180-degree rotation. The same ser and his residues enable hydration of (d) to (e).
The resolution to the dilemma is the finding that substrates attached to enzymes – which
are themselves chiral – can be converted to chiral compounds because they are attacked
selectively from one face of the substrate molecule. In the case of the aconitase reaction, the
intermediate cis-aconitate is fixed into its position on the enzyme by ionic attachment of the
carboxylates, and the positions of the enzymatic functional groups – the histidine and serine
residues and the Fe–S cluster (indicated as Fe/S in the diagram).
10.4.3 CIS-ACONITATE AS A KREBS CYCLE INTERMEDIATE
Early studies suggested that cis-aconitate was a bound intermediate of aconitase and not
released to solution. This adhesion would make the aconitase reaction a single conversion
with citrate as substrate and isocitrate as product. However, the mechanism requires a “flip”
of the intermediate cis-aconitate. The reason for this requirement is that the same activate
site and the same amino acid residues are used for dehydration and re-hydration reactions. It
is difficult to envision how the intermediate cis-aconitate could stay bound to the enzyme –
with multiple attachment points – and still flip without a release to solution.
A metabolic event resolves the issue. Cis-aconitate is a known substrate for a subsequent
enzyme in mitochondria as well as in bacteria. The reaction, catalyzed by a decarboxylase,
produces itaconate:
COO
H C H
C COO
CH2
Itaconate
Itaconate formation can be a significant pathway in macrophage cells during inflammation

(see the chapter addendum). From the standpoint of the aconitase reaction, it is clear that
cis-aconitate must diffuse from the enzyme surface in order to react in a separate metabolic
pathway. Therefore, aconitase catalyzes two distinct enzymatic reactions.
10.4.4 PROCHIRALITY
The realization that achiral molecules can nonetheless be converted to chiral ones also led
to a new concept: prochirality. A molecule with no chiral centers that can be directly con-
verted to a chiral molecule is known as a prochiral molecule. In the aconitase reaction, the
prochiral molecule is cis-aconitate because this is the species that is directly converted to
a chiral molecule. Virtually all prochiral molecules in biochemistry have a carbon engaged
in a double bond, that is, sp2-hybridized. While there are three carboxyl group attachment
sites in cis-aconitate, the requirement is not the number of groups attached to the enzyme,
but rather the unambiguous binding of a molecule to present it to enzymatic groups for a
stereospecific reaction.
To understand this point intuitively, consider the coffee cup in Figure 10.10a. There is a
plane of symmetry drawn through the cup in Figure 10.10b. If you grab this cup by the single
handle with your right hand (literally chiral!), you will be able to drink it only on the side fac-
ing you. Thus, the cup is prochiral, and the chiral binding entity is you! There is just one point
of attachment that establishes stereospecificity.
There are many other prochiral molecules in biochemical pathways. We have, in fact,
already encountered one: pyruvate. In its conversion to lactate, catalyzed by lactate DH,
pyruvate is converted to a chiral molecule, L-lactate (Figure 10.11). As is the usual case, the
prochiral center of pyruvate is sp2-hybridized.
FIGURE 10.10 Coffee Mug Prochirality. The coffee mug (a), if held by the right hand, will only
be sipped from the the side facing the viewer. The mug itself has a plane of symmetry, as shown
in (b).
FIGURE 10.11 Prochirality of Lactate DH. The lactate DH reaction product is exclusively
L-lactate.
10.4.5 THE R/S SYSTEM OF NOMENCLATURE
We have used the D/L system for assigning chiral centers, but this requires alignment with
the reference molecule glyceraldehyde. Clearly, comparison of glyceraldehyde with isocitrate,
COO
H C H
*
H C COO
HO C
* H
COO
Isocitrate
with two chiral centers, is ambiguous.

A different system to distinguish chiral molecules independent of a reference molecule,
called the RS System, can assign any number of chiral centers to any molecule. To apply
this system, consider a generalized chiral center with substituents labeled as a, b, c, and d
(Figure 10.12a). The two possible enantiomers are shown in this figure. We assume that the
substituents are in the order of their molecular weights so that a > b > c > d. The tetrahedron
is arranged so that the smallest – d – is behind the plane of the paper, and the other three are
in front of it. Next, imagine the three projecting substituents as points on a steering wheel.
Connect them by drawing an arc starting with the highest in molecular weight, from a to b
FIGURE 10.12 The RS System of Stereochemical Designation. (a) Enatiomers of a molecule

having a chiral center and four distinct subunits a,b,c, and d. (b) Ranking molecular weight from
highest (a) to lowest (d), the lowest is placed behind the plane of the paper, and the remaining
three projecting out as a “steering wheel”. Tracing in the clockwise direction is the R configura-
tion; tracing in the counterclockwise direction is the S configuration. (c) Stereochmeistry of 2R,
3S isocitrate.
to c. The two possible arcs are shown in Figure 10.12b. If the arc is in the clockwise direction,
the carbon center is defined as R (Latin, Rectus). If the arc is in the counterclockwise direc-
tion, the carbon center is defined as S (Latin, Sinister).
There are a few more rules needed to assign chiral centers in general. First, groups directly
attached to the chiral compound may be the same. In this case, we move further down the
chain and consider the next connected substituent. Secondly, a substituent may have mul-
tiple bonds. In this case, the atom across the double bond is counted twice (double bond) or
three times (triple bond).
We are now in a position to assign the two chiral centers of isocitrate, labeled as C3 and
C2 in Figure 10.12c. Each is separately drawn according to the rules outlined above. The full
designation of the molecule is 2R,3S isocitrate.
Like the DL system, there is no relationship between the direction of light rotation and
the assignment of R or S to a chiral center. The two systems are also similar in that they are
formal assignments that allow us to name specific molecular structures. While the RS sys-
tem is more general, it is less widely used in biochemistry than in other chemical specialties.
10.4.6 FLUOROACETATE POISONING
The fluorine derivative of acetyl CoA, fluoroacetate
F
H 2C COO
Fluoroacetate
is a natural product found in several plants from Australia, Africa, and South America. Its
toxicity stems from its conversion to the CoA thioester and subsequently to fluorocitrate
COO
H C H
HO C COO
F C H
COO
Fluorocitrate
using the citrate synthase reaction. Unlike citrate, fluorocitrate is a chiral compound. With
the fluoride attached, there are two chiral centers at positions 2 and 3. The inhibitory mol-
ecule is the 2R,3R isomer. The precise mechanism of inhibition is complex. Fluorocitrate can
be converted by aconitase to 4-hydroxy-trans-aconitate, which tightly binds aconitase and
inhibits the enzyme. Thus, fluorocitrate is a suicide substrate: the substrate analog employs
the catalytic machinery of the enzyme and produces a dead-end inhibitor rather than a prod-
uct. A second mode of action is the fluorocitrate inhibition of citrate transport from mito-
chondria to cytosol. Poisoning by fluoroacetate is thus indirect, involving both of the first
two steps of the Krebs cycle and displaying stereoselectivity; other isomers are not inhibitory.
The mode of inhibition is reversible, although the binding to the enzyme is very tight.
10.4.7 ISOCITRATE DH
In the isocitrate DH step, oxidation is tied to the release of CO2. Overall, the reaction is:

(10.3) Isocitrate + NAD+ ® a-Ketoglutarate + NADH + CO2
Like citrate synthase, this is a metabolically irreversible reaction. There is a separate isozyme
form in the mitochondrial matrix that uses NADP+ instead of NAD+. Eukaryotic cells also
have a third isozyme of isocitrate DH that uses NADP+ as cofactor, is located in the cytosol,
and achieves near-equilibrium.
The mechanism of Equation 10.3 involves oxidation to a chemically unstable β–ketoacid
intermediate followed by a decarboxylation, illustrated in Figure 10.13. The ketoacid is formed
in the first step as NAD+ accepts electrons from the hydride of the hydroxyl-containing car-
bon, in a manner identical to other dehydrogenases (e.g., glyceraldehyde phosphate DH).
Electron rearrangement leads to a loss of the carboxyl group from the middle position, pro-
ducing the enol form of α–ketoglutarate. The enol form is in equilibrium with the keto form,
shown as the last step of the reaction. A similar enol–keto equilibrium occurs with pyruvate.
FIGURE 10.13 Mechanism for lsocitrate Dehydrogenase. The ketoacid intermediate is formed
through the removal of a hydride from the alcohol of the substrate onto the acceptor, NAD+.
Next, the electron migration shown leads to loss of CO2.
10.4.8 α-KETOGLUTARATE DH COMPLEX
The other reaction that produces CO2 in the Krebs cycle is the α-ketoglutarate DH step:
(10.4) a-ketoglutarate + CoA + NAD+ ® succinyl-CoA + NADH + CO2
The overall reaction involves an enzyme complex similar to the pyruvate dehydrogenase
complex. The mechanisms for the two complexes are identical. They both use El, E2, and E3;
they both use thiamine-PP, lipoamide, and FAD in the same way. Both employ the swing-
ing arm mechanism. Indeed, the E2 portion of the complex is the same protein for both
complexes. The α-ketoglutarate DH complex is also a metabolically irreversible step and an
important regulatory site of the Krebs cycle, described in section 10.5.
10.4.9 SUCCINYL-COA SYNTHETASE
The only reaction of the Krebs cycle in which a high-energy phosphate is directly formed is
the succinyl-CoA synthetase step:

(10.5) Succinyl-S-CoA + GDP + Pi ® Succinate + GTP + CoA-SH
This metabolically irreversible reaction is unusual because it uses guanine nucleotides rather
than adenine nucleotides in many cells. Notable exceptions are the enzyme in mammalian
brain cells and plant cells, which use ADP/ATP in place of GDP/GTP.
The mechanism of the reaction (Figure 10.14) resembles others we have previously con-
sidered. Like the last step of the glyceraldehyde phosphate DH mechanism (Chapter 9), the
first step of this reaction is the displacement of a thioester by inorganic phosphate, forming
an acid phosphate. Like the phosphoglyceromutase reaction mechanism, the high-energy
phosphate is next transferred to a histidyl group of the enzyme, releasing succinate and
leaving the enzyme in a phosphorylated form. Finally, the enzyme-bound phosphoryl acts
as a nucleophile that attacks GDP, forming the final product, GTP, and regenerating the
enzyme in its original form. Succinyl-CoA synthetase (often called succinate thiokinase)
produces a high-energy phosphate compound (GTP) that can be directly utilized as an
energy intermediate. Like ATP formation in glycolysis, this process is known as substrate-
level phosphorylation.
FIGURE 10.14 Succinyl-CoA Synthetase. This mechanism is reminiscent of two previously

described ones. The first step is a phosphate displacement of a thioester, just like the terminal
step of glyceraldehyde phosphate DH. The next two are phosphoryl transfers to an enzyme
histidine residue, just like the phosphoglycerate mutase reaction.
10.4.10 SUCCINATE DH
Succinate DH catalyzes the conversion of succinate to fumarate, and is the only membrane-
bound enzyme of the Krebs cycle:

(10.6) Succinate + UQ ® fumarate + UQH 2
The mobile cofactor ubiquinone, introduced in Chapter 7, accepts electrons from succinate
in an indirect way, since succinate is water-soluble and UQ is immersed within the lipid
portion of the inner membrane bilayer (Figure 10.15). Succinate DH is an enzyme complex,
with several proteins involved in the overall reaction. Succinate DH, like the pyruvate DH
and α-ketoglutarate DH complexes, consists of multiple proteins that shuttle intermediates
within the complex without connection to any other metabolic step. UQ reacts with a differ-
ent protein face than succinate. The succinate DH complex is also involved in mitochondrial
electron transport; further details are presented in Chapter 11.
Succinate DH is also notable as the step inhibited in the classic experiments of Krebs,
based on the structural similarity between succinate and malonate.
H3C
COO
CH2 COO HN O
CH2 CH2
COO COO
Succinate Malonate Fenfuram

FIGURE 10.15 Succinate DH. Succinate reacts with the enzyme complex succinate DH in the
matrix space, donates electrons to the complex that are ultimately accepted by UQ within the
inner mitochondrial membrane. The product fumarate appears in the matrix space, and the
reduced cofactor UQH2 is within the membrane.
FIGURE 10.16 Fumarase. This hydration of fumarate to malate is similar to the aconitase
reaction (Figure 9.8) in that fumarase is prochiral, has an Fe-S cluster, and produces exclusively
L-malate.
Malonate can bind the enzyme in a similar way to succinate, but there can be no double bond
formation with just the single methylene group between the carboxylates. The structure of
a more modern succinate DH inhibitor, fenfuram, is also shown. There is little structural
resemblance to the substrate; this and many other inhibitors of the enzyme are designed by
modeling the X-Ray structure of the enzyme. These inhibitors are valuable as plant fungicides.
10.4.11 FUMARASE
The next reaction in the Krebs cycle is the hydration of fumarate to malate, catalyzed by
the near-equilibrium enzyme fumarase. The reaction adds water across the carbon-carbon
double bond of fumarate (Figure 10.16). Essentially, the reaction is the same as the aconitase
reaction converting cis-aconitate to isocitrate. There is an Fe/S center involved in binding the
substrate and coordinating the water molecule for nucleophilic attack. The prochiral sub-
strate fumarate is converted into the chiral product L-malate by the fixed attachment to the
enzyme surface (through the two carboxyl groups) and the specific addition of the hydroxyl
group from only one side of the substrate face.
The fumarase reaction is catalyzed at near-equilibrium, and thus does not represent a
potential control point for the Krebs cycle.
10.4.12 MALATE DH
The final step in the Krebs cycle is catalyzed by malate DH, another near-equilibrium reac-
tion (Figure 10.17). The mechanism involves hydride transfer from the C–H bond of the
FIGURE 10.17 Malate DH. The malate DH reaction is yet another redox reaction involving
hydride transfer. As it is near-equilibrium, the reverse reaction also produces a new chiral center.
substrate (L-malate) to the NAD+ ring, as in the other dehydrogenase reactions. The reac-
tion product oxaloacetate is achiral, but when the reaction runs in the reverse direction,
which happens in pathways other than the Krebs cycle, the product is always the L-isomer
of malate. Thus, we have yet another case of a reaction that produces a chiral molecule from
a prochiral one.
10.5 ENERGY BALANCE
Estimating the total ATP production from the oxidation of acetyl-CoA by the Krebs cycle
requires assumptions about how much energy can be extracted by the energy cofactors
(Box 10.3). The approximate equivalents for calculations are:
NADH: 3 ATP
UQH2: 2 ATP
GTP: 1 ATP
With these assumptions, the three NAD+-linked dehydrogenase reactions produce 9 ATP;
the single UQH2-generating step catalyzed by succinate DH produces 2 ATP; and the succi-
nyl-CoA synthetase reaction produces 1 GTP, roughly equivalent to 1 ATP. Indeed, in many
cells, the enzyme uses ADP as a substrate, producing ATP directly. The total is 12 ATP per
acetyl-CoA oxidized. How NADH and UQH2 oxidations can produce ATP requires a study
of the electron transport chain of the mitochondria. However, it is already clear that the
Krebs cycle produces prodigious amounts of ATP when glucose is fully oxidized to CO2. A
single glucose molecule produces 38 ATP via complete oxidation, compared to 2 ATP using
glycolysis alone (Table 10.2).
Box 10.3 Estimating ATP Energy Equivalents
The values stated in this text are the “classical” ones that have been used for many years:
3 ATPs for NADH oxidation and 2 ATPs for UQH2 oxidation. For metabolic analysis in
nutrition and medicine, these conversion factors are universally accepted. However, it is
known that these values are not precise. The use of exact integer values given above is a
throwback to an era when it was believed that ATP was synthesized locally at three sepa-
rate sites in mitochondria. When succinate was oxidized, it was believed that it skipped
“site 1” and produced only 2 ATPs. Once it became clear that a proton gradient was the
energy intermediate, fractional values – which had been observed in many labs – became
a possibility. Many experimental studies have arrived at values of 2.5 and 1.5, for NADH
and UQH2, respectively. Also, the amount of ATP formed from GTP must be less than unity,
because the efficiency cannot be 100%. These considerations show that the estimates
of ATP equivalents, while useful, are inexact. The lack of certainty surrounding the later
experiments and the fact that the values are usually taken to compare pathways is why
the whole numbers presented in the text are widely used.
228 10.6 Regulation
TABLE 10.2 Energy Production by the Full Oxidation of Glucose

Metabolic Sequence Energy Cofactor Equivalents of ATP
Glucose → Pyruvate 2 ATP 2
2 NADH 6
2 Pyruvate → 2 Acetyl-CoA 2 NADH 6
2 Acetyl-CoA → 2 CO2 6 NADH 18
2 QH2 4
1 GTP 2
Overall Glucose → 6 CO2 38
10.6 REGULATION
There are two levels of regulation of the Krebs cycle: the supply of the pathway substrate ace-
tyl-CoA, and control of the enzymes of the cycle itself. We have emphasized the production
of acetyl-CoA from pyruvate, which in turn arises from glycolysis. However, as discussed
elsewhere in this text, all major nutrients – fats, carbohydrates, and proteins – are converted
into acetyl-CoA for oxidation by the Krebs cycle. Thus, the Krebs cycle is the final common
route for the complete oxidation of foods into CO2.
Regulation of pyruvate to acetyl-CoA – that is, control of the pyruvate DH complex – is
primarily exerted by phosphorylation and dephosphorylation of the E1 component. The lat-
ter events require two additional components of the complex itself, a protein kinase and
protein phosphatase (Table 10.1). Control of those enzymes is illustrated in Figure 10.18. The
pyruvate dehydrogenase kinase (PDH kinase) that catalyzes phosphorylation of E1 is alloste-
rically inhibited by pyruvate and allosterically stimulated by acetyl-CoA. Because conversion
to the phosphorylated form of E1 causes its inactivation, pyruvate stimulates overall pyruvate
dehydrogenase complex activity, and acetyl-CoA inhibits it. Thus, the substrate and product
of the complex itself also serve as allosteric regulators of the kinase. The protein phosphatase
activity of E1 (PDH phosphatase) is stimulated by Ca2+ ions, leading to dephosphorylation of
E1 and stimulation of the overall complex.
Aside from the regulation of the supply of acetyl-CoA to the Krebs cycle, several steps
in the pathway itself are metabolically irreversible and thus potentially regulatory. Citrate
synthase, isocitrate DH, α-ketoglutarate DH, succinyl CoA synthetase, and succinate DH all
FIGURE 10.18 Regulation of the POH Complex. E1 (the PDH component) is phosphorylated
and inactivated in a reaction catalyzed by PDH kinase. PDH kinase is inhibited by pyruvate and
stimulated by acetyl-CoA.The protein phosphatase that catalyzes dephosphorylation of E1-P is
stimulated by Ca2+.
fit this classification. Of these, isocitrate DH and α-ketoglutarate DH can be stimulated by an

increase in the concentration of Ca2+ ions. Control of the other enzymes is usually related to
control of flow through different reactions connected to the Krebs cycle.
There is another regulatory circuit that operates between the Krebs cycle and glycolysis.
As the rate of the Krebs cycle increases, so do the concentrations of its intermediates. An
increase in citrate concentration in mitochondria leads to increased cytosolic citrate, as there
is a transporter for citrate in the mitochondrial inner membrane. An increased citrate con-
centration in the cytosol decreases phosphofructokinase activity and hence glycolysis. The
underlying observation that glycolysis is diminished in aerobic metabolism was made in the
19th century and is known as the Pasteur effect.
10.7 KREBS CYCLE AS A SECOND CROSSROAD

OF METABOLIC PATHWAYS
We have just considered the reactions of the Krebs cycle as a complete pathway for the oxida-
tion of acetyl-CoA to CO2. The Krebs cycle can also be viewed as a metabolic hub. Each inter-
mediate in the Krebs cycle is part of another metabolic pathway. Some of those pathways
are major routes in metabolism. For example, fatty acid synthesis (Chapter 14) uses citrate
from the Krebs cycle to supply the carbon for lipids. Citrate removed from the cycle must
be replaced; thus, separate reactions must provide input to the cycle. The term for a reaction
that directly provides carbon input to the Krebs cycle is anaplerotic, or “refilling”.
Some amino acids can be converted to Krebs cycle intermediates by the process of trans-
amination. Comparing the structure of the amino acid glutamate with 2-ketoglutarate
COO COO
CH2 CH2
CH2 CH2
HC NH2 C O
COO COO
Glutamate α-Ketoglutarate
you may notice that the two are similar except for the amine in one case and the keto group
in the other. Similarly, aspartate can be viewed as an amine analog of the Krebs cycle inter-
mediate oxaloacetate:
COO COO
H2C CH2
HC NH3 C O
COO COO
Aspartate Oxaloacetate
The Krebs cycle also connects to pathways that are more elaborate and not considered in
detail in this text. For example, heme biosynthesis is a pathway stemming from succinyl-
CoA. Two further examples are examined in the chapter addendum: the glyoxylate cycle,
found in bacteria and plants; and the itaconate pathway, found in organisms from bacteria
to humans. Like an airline hub that connects numerous sub-stations, the Krebs cycle inter-
mediates are connected to separate pathways in the cell. It is always important to remember
that, as a distinct pathway, the Krebs cycle has just one substrate, acetyl-CoA, and one prod-
uct, CO2.
230 Summary
SUMMARY
All major nutrients – carbohydrates, lipids, and proteins – are oxidized first to acetyl-CoA
and then to CO2 through the Krebs cycle. When the precursor is a carbohydrate, pyruvate
is the source of acetyl-CoA, and the pyruvate dehydrogenase complex catalyzes the conver-
sion. The pyruvate DH complex has three separate enzymes and five cofactors: thiamine
pyrophosphate, lipoic acid, CoA, FAD, and NAD+; the latter two in their reduced forms are
FADH2 and NADH. In the first step of the Krebs cycle, acetyl-CoA condenses with oxaloac-
etate to form citrate, a six-carbon intermediate. Next, aconitase catalyzes the conversion of
citrate to prochiral cis-aconitate and to chiral isocitrate. Prochirality is the conversion of an
achiral substrate to a chiral product. Isocitrate loses CO2 in the next step, a reaction cata-
lyzed by isocitrate dehydrogenase, thus forming the five-carbon molecule α–ketoglutarate.
This is followed by the other decarboxylation reaction of the Krebs cycle, catalyzed by the
α–ketoglutarate DH complex. This complex is closely analogous to the pyruvate DH com-
plex, because it has the same three types of enzymes, the same five cofactors, and the same
mechanism. Next, succinyl-CoA and GDP are converted to succinate and GTP, catalyzed
by succinyl CoA synthetase. Succinate is converted to fumarate via succinate DH, the only
membrane-bound enzyme of the cycle. Succinate DH funnels reducing equivalents into the
lipid-soluble mobile cofactor ubiquinone. The fumarase catalyzed hydration of fumarate pro-
duces another new chiral center in L-malate, another example of prochiral discrimination
by enzymes. The final step is the oxidation of malate to oxaloacetate, with the formation of
NADH. Overall, the Krebs cycle produces 12 ATP molecules for each input acetyl-CoA. The
Krebs cycle is regulated in part by the concentration of acetyl-CoA, the pathway substrate.
The pyruvate dehydrogenase complex is regulated by phosphorylation (inhibition) and by
mitochondrial Ca2+ concentration (stimulation). Ca2+ ions also directly stimulate the α–keto-
glutarate DH complex. Aside from its role as the final oxidation pathway in cells, the Krebs
cycle serves as a metabolic hub; each enzymatic step is utilized as a part of other pathways
in the cell.
REVIEW QUESTIONS
1. One consequence of a cyclic pathway is that its intermediates are in very low, catalytic
amounts. However, in the linear pathway of glycolysis, the intermediates are also in
very low concentrations. How is a cyclic pathway fundamentally different?
2. The Krebs cycle has two basic strategies for oxidizing molecules and extracting elec-
trons as mobile carriers. What are they?
3. How does fluoroacetate act as a poison?
4. Explain the notion of prochirality and where it is significant in the Krebs cycle.
5. Which carbon of acetyl-CoA attaches to oxaloacetate in the citrate synthase reaction?
6. Which step of the Krebs cycle is called substrate-level phosphorylation? What step
does this correlate to in glycolysis?
7. Which steps of the Krebs cycle produce CO2? Which produce NADH?
8. Which reaction of the Krebs cycle has the same mechanism as the pyruvate dehydro-
genase complex?
9. Malonate is chemically similar to succinate, as both are dicarboxylic acids with inter-
vening methyl subunits. Why can’t malonate also be a substrate of succinate DH?
What type of inhibition would you predict for malonate?
CHAPTER 10 ADDENDUM: CYCLIC PATHWAYS

CONNECTED TO THE KREBS CYCLE
Two cyclic pathways that intersect with intermediates of the Krebs cycle are the Glyoxylate
cycle and the Itaconate Pathway. Each is present in microorganisms and plants; the Itaconate
Pathway has recently been found in human macrophages.
1. GLYOXYLATE CYCLE
The glyoxylate cycle is a means of converting acetyl-CoA to glucose, a pathway that does not
occur in animals but does in some bacteria, yeast, and plants. The bacterial route is shown in
Figure CA10.1 as a bypass of the Krebs cycle.
Two enzymes unique to the glyoxylate pathway are boxed in the figure: isocitrate lyase,
which catalyzes hydrolysis of isocitrate to glyoxylate and succinate; and malate synthase,
which catalyzes condensation of acetyl-CoA with glyoxylate to form malate. Note the paral-
lels of these enzymes to citrate lyase and citrate synthase, respectively.
When the two enzymes are induced in bacteria, they enable a net conversion of acetyl-
CoA to glucose. One acetyl-CoA condenses with oxaloacetate to form citrate; a second
acetyl-CoA condenses with glyoxylate to form malate. This malate can then be converted to
glucose.
An example of the medical significance of this pathway is the case of the tuberculosis
bacteria, which survive metabolic isolation resulting from phagocytosis in the lysosome by
induction of the glyoxylate cycle. This greatly improves bacterial metabolic flexibility, as glu-
cose can now be formed from acetyl-CoA. The glyoxylate cycle enables the bacteria to resist
the innate immune system. The glyoxylate cycle is also present in yeast and plants; how-
ever, the pathway is more complicated in these organisms. The two specific glyoxylate cycle
enzymes exist exclusively in a separate organelle, the glyoxysome. Thus, in these organisms,
the pathway spans the glyoxysome, the cytosol, and the mitochondria.
2. ITACONATE PATHWAY
Decarboxylation of cis-aconitate is the first step in the pathway outlined in Figure CA10.2.
The five-carbon product molecule is itaconate, which is unusually reactive due to its terminal
alkene. Some enzymes are inactivated upon binding this molecule, including the isocitrate
FIGURE CA10.1 The Glyoxylate Cycle. The two enzymes shown in shaded boxes, isocitrate
lyase and malate synthase, catalyze a bypass of the Krebs cycle, avoiding the decarboxylation
steps and producing net four-carbon intermediates.
232 Key Terms
FIGURE CA10.2 The Itaconate pathway. The first intermediate of the pathway is itaconate,
the result of decarboxylation of the Krebs cycle intermediate cis-aconitate. Itaconate can accu-
mulate in some cells of the immune system, or be further metabolized as shown to pyruvate and
acetyl-CoA.
DH step of the Krebs cycle. In human macrophages, itaconate can accumulate to such high
levels that it becomes the most abundant intermediate in the cell and can serve to inacti-
vate proteins leading to pro-inflammatory cytokines. In this way, itaconate may dampen the
inflammation that is initially triggered in the immune response. Earlier studies have shown
that itaconate may also be involved as an immune cell defense against bacteria residing in
the lung. As discussed above, the glyoxylate cycle potentiates tuberculosis bacteria’s survival.
Itaconate secreted by macrophages can enter these bacteria to some extent, inactivate isoci-
trate DH, thereby inhibiting the glyoxylate cycle.
More recently, itaconyl-CoA has been shown to bind as a substrate mimic to the enzyme
methylmalonyl mutase (see Chapter 14), a step in odd chain fatty acid oxidation. This step
requires vitamin B12 (cobalamin); when itaconyl-CoA binds, it leads to the inactivation of
B12. Thus the enhanced itaconate pathway in hyper-immunostimulation may deplete vita-
min B12. There is also evidence that accumulation of itaconate through induction of the
aconitate decarboxylase may occur more widely in cells involved in innate immunity, includ-
ing monocytes and dendritic cells, as well as in some epithelia.
KEY TERMS
anaplerotic
citric acid cycle
enzyme complex
inner mitochondrial membrane
Krebs cycle
matrix space
outer mitochondrial membrane
Pasteur effect
porin
prochiral center
pyruvate dehydrogenase complex
pyruvate transporter
repetition–variation
secondary metabolism
substrate level phosphorylation
suicide substrate
tricarboxylic acid cycle
BIBLIOGRAPHY
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1818 (2012) 1438–1443. A history of the discovery of the outer membrane channel by two of its
research pioneers.
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Metabolism. Glia 21 (1997) 106–113.
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Hybrids as Potent Succinate Dehydrogenase Inhibitors. Bioorganic Chemistry 73 (2017) 76–82.
E.A. Newsholme, A.R. Leech. Biochemistry for the Medical Sciences. Wiley, New York. 1986.
R.S. Ochs, T.T. Talele. Revisiting Prochirality. Biochimie 170 (2020) 1–8. A proposal to explain prochi-
rality in enzyme action.
O.E. Owen, S.C. Kalhan, R.W. Hanson. The Key Role of Anaplerosis and Cataplerosis for Citric Acid
Cycle Function. Biological Chemistry 277 (2002) 30409–30412.
W.C. Stallings, C.T. Monti, J.F. Belvedere, R.K. Preston, J.P. Glusker. Absolute Configuration of the
Isomer of Fluorocitrate That Inhibits Aconitase. Archives of Biochemistry and Biophysics 203
(1980) 65–72.
M. Steiger, M. Blumhoff, D. Mattanovich, M. Sauer. Biochemistry of Microbial Itaconic Acid
Production. Frontiers in Microbiology 4 (2013).
W.H. Tong, T.A. Rouault. Metabolic Regulation of Citrate and Iron by Aconitases: Role of Iron-Sulfur
Cluster Biogenesis. Biometals 20 (2007) 549–564.
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Molecular Immunology (2020). Review indicating occurance of itaconate pathway in immune
cells and beyond.
X.H. Yu, D.W. Zhang, X.L. Zheng, C.K. Tang. Itaconate: An Emerging Determinant of Inflammation in
Activated Macrophages. Immunology and Cell Biology 97 (2019) 134–141.
Oxidative
Phosphorylation 11
Electrons from the Krebs cycle are captured as two mobile carriers: NADH and UQH2.
The pathway that oxidizes these compounds is called oxidative phosphorylation, and it is
responsible for most of the ATP produced in cells. A unique feature of oxidative phosphory-
lation is a proton gradient that acts as the energy intermediate. The mechanism is known as
the chemiosmotic hypothesis, initially proposed by Peter Mitchell in the early 1960s. At
first a novel hypothesis, this mechanism is now universally accepted not only for mitochon-
drial energy production but for photosynthesis as described in Chapter 12.
11.1 THE PHENOMENON
The term oxidative phosphorylation refers to two processes: oxidation involving electron
flow, and phosphorylation, the synthesis of ATP from ADP and Pi. In isolated mitochondria,
as in intact cells, these processes are coupled. A simple experiment with mitochondria shows
this coupling directly: adding ADP to the preparation causes the oxidation of substrates, the
consumption of oxygen, and the formation of ATP. NADH is the major electron donor, while
UQH2 enters as an intermediate in the process. The overall equation for electron flow is:

(11.1) NADH ® UQ ® O2
NADH is oxidized to NAD+, consistent with its role as a mobile cofactor, and O2 is reduced to
water. Thus, there is a continuous consumption of O2 and production of H2O. Another kind
of coupling occurs in this process, closely related to the first: a coupling of electron and pro-
ton movement. The movement of electrons from NADH to O2 takes place exclusively within
the inner membrane of the mitochondria. Electrons are passed from one electron carrier to
another in a series of steps collectively called the electron transport chain. As electrons
move through the membrane, protons move across the membrane, from the interior of the
mitochondrial matrix to the outside of this inner membrane (Figure 11.1). The movement of
protons is vectorial because it has both a magnitude (the number of protons transported)
and a direction (from the matrix to the cytosol). We will get a sense of how this coupling
works by considering some details of the molecules involved in supporting this process. The
movement of protons across the inner membrane leaves the inner membrane space nega-
tively charged relative to the outside of the matrix. There is also a concentration gradient
of protons, with greater proton accumulation outside the matrix. Hence, there is both an
electrical gradient (charge separation) and a chemical gradient (differences in concentration)
across the membrane. The proton gradient itself is the energy intermediate of the entire
process. The re-entry of protons occurs through a channel linked to ATP synthesis. We can
summarize this overview by saying that oxidation is the stepwise removal of electrons from
substrates that ultimately reduce oxygen to water and that it creates a proton gradient as it
does so. Phosphorylation is the process of importing protons back into the mitochondrial
matrix, forming ATP from its precursors, ADP and Pi.
235
236 11.2 Mitochondrial Inner Membrane
FIGURE 11.1 Vectorial transport. As electrons move through the membrane, protons move
across it.
FIGURE 11.2 A mitochondrion under the electron microscope. (Source: Don W. Fawcett/
Photo Researchers, Inc., with permission)
11.2 MITOCHONDRIAL INNER MEMBRANE
An electron micrograph of a typical mitochondrion is shown in Figure 11.2. The outer

membrane is evident as the outer perimeter of the organelle. The inner membrane features
infoldings, which increase membrane surface area. A sketch of the essential topology of
mitochondria is presented as Figure 11.3a. As with the entry of pyruvate into the mitochon-
dria, large pores (porin or VDAC, described in Chapter 10) exist in the outer membrane,
rendering it permeable to molecules up to 10,000 daltons. It is experimentally possible
to strip off the outer membrane of isolated mitochondria and reproduce all of the events
described in this chapter. Hence, only the inner membrane is critical for oxidative phos-
phorylation. It also follows that the space outside the inner membrane, the inner mem-
brane space, is equivalent to the cytosol for small molecules under 10,000 daltons. As a
result, our view of mitochondrial topology can be simplified to the functional picture pre-
sented as Figure 11.3b, in which the inner membrane – usually referred to simply as “the
membrane” divides the matrix from the cytosol. The inner membrane contains a relatively
high concentration of proteins, which includes exchangers like the pyruvate transporter
and several protein complexes embedded in the membrane that conduct the flow of protons
Chapter 11 – Oxidative Phosphorylation 237
FIGURE 11.3 Spaces in mitochondria. (a) Diagram mapping membranes and spaces. (b)
Functional essence of mitochondrial spaces.
and electrons. We can classify carriers for the electron transport chain into three groups:
molecules that carry electrons, protons, or both.
11.3 CARRIERS OF ELECTRONS, PROTONS, OR BOTH
11.3.1 CARRIERS OF ELECTRONS
Metals are pure electron carriers; the most common of these is iron, with copper a close sec-
ond. The metal ions of the electron transport chain exist in a chelate form, either as a heme or
as a complex with sulfur, such as the Fe-S complex. The heme iron in hemoglobin and the Fe-S
clusters in aconitase and fumarase are not redox-active. Instead, in these proteins, the iron is
exclusively in the Fe2+ state. However, as electron carriers in oxidative phosphorylation, metals
rapidly undergo changes in redox state, such as Fe2+ alternating with its oxidized form, Fe3+.
Those compounds that are pure electron carriers always mediate single-electron transfers.
11.3.2 CARRIERS OF PROTONS
We have previously encountered pure carriers of protons in another guise: as acids or

bases. Thus, aspartyl residues of proteins are proton carriers, as are bases such as histidyl
238 11.4 Membrane-Bound Complexes
residues of proteins. These side groups play an essential role in establishing the proton
gradient.
11.3.3 CARRIERS OF BOTH ELECTRONS AND PROTONS
The major carriers of both electrons and protons are the mobile cofactors NADH and UQH 2,
and the prosthetic groups FADH2 and FMNH2 (Chapter 7). NADH transfers an electron pair
as a hydride ion (H−). By contrast, UQH2 transfers single electrons only, in two distinct steps.
The process, illustrated in Figure 7.3, is fully reversible. UQ accepts one electron to form
intermediate UQ●−, a radical ion.
H3CO CH3
H3CO 10
UQ•–
The unpaired electron (making the structure a radical) is delocalized through the ring.
However, the negative charge on the other oxygen is localized. As the rest of the molecule
is hydrophobic, UQ●− is an amphiphile, with consequences for the mechanisms of electron
transfer.
The flavin cofactors (Chapter 7) are unique in that they can transfer electrons either
one at a time or two at a time. In the single-electron transfer mode, there is a radical
intermediate, stabilized by the aromaticity of the ring. Alternatively, flavins can undergo
a two-electron, hydride ion transfer. It is this ability that makes the flavin cofactors (FAD
and FMN have identical redox properties) transducers between the obligate two-electron
exchange with the cofactor NADH and all of the other cofactors that transfer one electron
at a time.
11.4 MEMBRANE-BOUND COMPLEXES
Five membrane-bound protein complexes participate in oxidative phosphorylation. These

can be viewed as islands within the membrane sea that only communicate with each other
indirectly. For example, the mobile cofactor NADH can donate electrons and protons to
Complex I (Figure 11.4). This complex releases protons to the cytosol and transfers its elec-
trons to UQ, forming UQH2. UQ and UQH2 are mobile cofactors confined to the hydropho-
bic interior of the membrane. UQH2 donates its electrons to complex III and releases protons
to the cytosol. The electrons of complex III reduce cytochrome c, a mobile cofactor that
slides along the cytosolic surface of the membrane and donates its electrons to complex IV.
Finally, electrons from complex IV react with O2 to form H2O.
The pathway just described traces the route electrons follow from NADH to O2 , and
it involves complexes I, III, and IV, and the intermediary mobile cofactors UQH 2 and
cytochrome c. Complex II also is known as succinate dehydrogenase, an enzyme that
catalyzes a step in the Krebs cycle. Electrons from succinate enter this complex and they
leave as UQH 2 , so that UQ collects electrons from complexes I and II. As we will appre-
ciate later, other complexes in the mitochondrial membrane also form UQH 2 , so this
cofactor is a point of common intersection. Together, the first four complexes account
for electrons movement in oxidative phosphorylation as well as the generation of the
proton gradient.
H+
H+
UQH2
Cytosol
UQ Complex
III
Complex
I
Cyt c ox
H+
H+
Matrix Cyt c red

NADH NAD+
1
O2 + 2 H+
H+ 2
ADP + Pi
H2O Complex
IV
H+ ATP
Complex H+
V H+
Fumarate Succinate
Complex Inner mitochondrial

UQ
II membrane
UQH2
FIGURE 11.4 Schematic of oxidative phosphorylation. Oxidative phosphorylation is illustrated

as a function of five complexes and the communicating cofactors. Protons are the intermediate
between redox flows (in complexes I, III, and IV) and ATP synthesis (complex V). There are three
types of mobile cofactors. NADH is water-soluble, UQ is soluble in the hydrophobic interior of the
membrane, and cytochrome c moves along the outer leaflet of the membrane.
Complex V uses the proton gradient to form ATP (Figure 11.4). As protons flow down
the gradient, complex V synthesizes ATP. We next consider the processes of the electron
pathway and proton circuits individually before we directly examine the operation of the
complexes.
11.5 THE ELECTROCHEMICAL CELL AND THE MITOCHONDRIA
As we observed in Chapter 8, the electrochemical cell is essentially a battery. The classical

Zn/Cu cell developed there is presented here as Figure 11.5a. It is possible to represent that
cell more schematically, as shown in Figure 11.5b. The wire has become simply a line for
electron flow, and the salt bridge a tube that connects ions. The direction of removal (Zn) or
addition (Cu) of ions is indicated. We can represent the oxidation of NADH and the reduc-
tion of O2 as an electrochemical cell by taking the symbolic representation even further, as in
Figure 11.5c. Just as before, electrons travel from NADH through a symbolic wire and end up
at the site of oxygen reduction. Meanwhile, protons are moved through a symbolic salt bridge
and are used to produce the hydrogens of water.
It was the genius of Peter Mitchell to envision that mitochondria are much like an elec-
trochemical cell, except that the roles of electron and ion movement must be swapped. Since
the electrochemical representation of the NADH oxidation coupled to O2 oxidation has been
made fully symbolic as in Figure 11.5c, this is readily accomplished. Figure 11.6a shows the
240 11.6 Electron Pathways
FIGURE 11.5 NADH → O2 as an electrochemical cell. The cell in (a) produces an electrical
potential as electrons move from the Zn to the Cu electrode and ions move from the Cu2+ solu-
tion to the Zn2+ solution; (b) symbolizes the electrochemical cell; and (c) NADH oxidation linked
with O2 reduction is drawn as an electrochemical cell.
FIGURE 11.6 Protonmotive representation of mitochondrial energy production. (a) Reversing

the ion and electron movements of the electrochemical cell of Figure 11.5c produces a proton-
motive cell. (b) Mapping the protonmotive cell onto a mitochondrion.
oxidation and reduction as before, except that now protons move through the “wire” and
electrons through the “salt bridge”. The scheme of flows can be mapped in a diagram more
closely resembling mitochondria, shown as Figure 11.6b. The electrons move through a “salt
bridge” which is identified as the mitochondrial inner membrane. Protons move through a
“wire” which corresponds to passageways that cross the membrane.
11.6 ELECTRON PATHWAYS
11.6.1 SEQUENCE OF ELECTRON FLOW
The pathway of connected redox reactions that comprises the respiratory chain was derived
from studies of oxygen consumption by mitochondria in response to inhibitors of known
specificity. The sequence is presented as Figure 11.7a. NADH passes electrons through
Complex I, reduces UQ, which in turn passes electrons to Complex III. Electrons from
Complex III are accepted by cytochrome c, which in turn reduces Complex IV. Finally, elec-
trons from Complex IV are used to reduce oxygen to water. Notice that succinate enters the
respiratory chain as a branch point by passing electrons to Complex II to reduce UQ.
The experiments elucidating the order of this chain were based on selective inhibitors of
the respiratory complexes. Measurements of the redox states of the complexes and the mobile
cofactors NADH, UQ, and cytochrome c, use specific visible light absorption bands of these
molecules. Consider, for example, the effect of cyanide. Its presence causes the reduction of
all components from NADH to cytochromes of Complex IV. Thus, cyanide must inhibit a
step very close to the oxygen side of the chain. On the other hand, rotenone (widely used as a
FIGURE 11.7 Sequence of electron transport. (a) The order of electron movement was experi-
mentally determined using inhibitors of each complex. (b) Structures of rotenone, malonate, and
antimycin A.
rat poison) causes a reduction only of NADH and oxidation of all of the other components as
oxygen reaction at Complex IV pulls all of the electrons from the chain into water formation.
Inhibition sites are indicated in Figure 11.7a; structures are shown in Figure 11.7b.
Malonate was the inhibitor used by Krebs in the original formulation of the Krebs cycle; in
that pathway, it is known as succinate DH. More potent inhibitors of Complex II are now
available (Box 11.1). Antimycin is a classical and still widely used inhibitor selective for
Complex III. Cyanide binds the site occupied by oxygen in Complex IV. Any of the inhibitors
of the respiratory chain can prove fatal to organisms that rely on oxidative metabolism.
11.6.2 ENERGETICS OF ELECTRON FLOW
In Chapter 9, we found that pathway energetics can be displayed as a plot of free energy ver-
sus pathway intermediates for the case of glycolysis. We can relate the sequence of electron
flow through the respiratory chain to their standard redox potentials, which is directly pro-
portional to free energy (Figure 11.8).
The redox (midpoint) potentials for the cofactors of the respiratory chain are presented
in Table 11.1. They form the basis of the placement of the reactions drawn in the pathway
progress diagram of Figure 11.8. The path is one of a steadily oxidizing flow. NADH can be
considered to be the pathway substrate because most of the electrons of metabolic pathways
are funneled into NADH. H2O is the common termination point and is thus the pathway
242 11.6 Electron Pathways
Box 11.1 A New Class of Complex II Inhibitors
Complex II is distinct from the other three mitochondrial complexes because it does not
contribute to the proton gradient. Genetically, it is separate because, unlike the other
four complexes, it is coded by DNA entirely from the cell nucleus rather than having at
least some protein components synthesized by mitochondrial DNA. Malonate is a classic
inhibitor that was used to help elucidate the Krebs cycle. However, it is less potent and
less selective than inhibitors of the other complexes. Recently, a series of compounds was
reported that are structural analogs of ubiquinone, called “atpenins”, such as atpenin A4.
Cl
H3C
CH3
H3 C
OH
O
H3C O
O N O
H
H 3C
Aptenin4
Atpenins, originally isolated from a fungus (genus penicillium), ironically have antifungal
activity. Compared to malonate, atpenins are over 1000-fold more potent inhibitors of
complex II.
–400
NADH
Complex I
0 Succinate Q
Complex III
E 0 (mV)
Cytochrome c
400
Complex IV
800 O2
Pathway progress
FIGURE 11.8 Redox sequence of electron flow. The sequence of electron transport is plotted
against the standard reduction potential. Three changes in reduction potential, for complexes
I, Ill, and IV, are large and contribute to energy capture. Free energy is directly proportional to
changes in redox potential.
product. It may seem surprising that these redox potentials – which are standard state
values – are used for the actual redox reactions. For most metabolic pathways, standard
thermodynamic values bear no relation to the actual ones; this was underscored in the study
of energetics of glycolysis shown in Figure 9.27. However, it is a reasonable approximation
to use standard values for the respiratory chain components. While the concentrations of
TABLE 11.1 Reduction Potentials

for the Components of the
Mitochondrial Respiratory Chain
Half Reaction E0 (mV)
NAD+ → NADH −320
Fumarate → Succinate −30
UQ → UQH2 −40
Cyt. c (ox) → Cyt. c (red) 300
½ O2 → H2O 820
TABLE 11.2 Reduction Potentials and Standard

Free Energies for Complexes I through IV
0
Complex E rxn (mV) ΔG0 (kJ/mol)
I: NADH Oxidase 360 −70
II: Succinate DH 10 ~0
III: UQH2 Oxidase 190 −37
IV: Cytochrome c Oxidase 600 −116
the respiratory chain components are not 1 M (standard conditions), they are in nearly 1:1
ratios; the redox components are mostly in complexes. Because the values on both sides of
the equation are similar, they cancel in the ratio and mimic standard state concentrations.
Because the pH is set to 7.0 (biochemical standard state) rather than 0 (chemical standard),
[H+] is also near its standard state. Finally, the ratio of NAD+ to NADH in the mitochondrial
matrix happens to have a value of about 1 in the mitochondrial matrix. In the cytosol, NAD+/
NADH is two orders of magnitude higher.
In a further analogy to the diagram of energetics of glycolysis (Figure 9.27), we can esti-
mate the change in redox potential for Complexes I through IV as illustrated by the length
of the vertical arrows in Figure 11.8. There is a substantial change in redox potential for
complexes I, III, and IV and nearly zero for Complex II. We can convert the change in redox
potential to the change in standard free energy using the equation developed in Chapter 8:

(11.2) DGo = -nFEorxn
which relates the redox potential for the reaction (which can be taken from the change in half
reactions, represented by the bars in Figure 11.8), with the standard free energy change. The
values for the complexes are listed in Table 11.2.
Experimentally, the free energy change (ΔG) of ATP synthesis has a value of approxi-
mately 44 kJ/mol. If we compare this with the ΔG0 values in Table 11.2, we would conclude
that Complexes I and IV have a free energy value sufficient to drive ATP synthesis, Complex
III is somewhat low, and that Complex II cannot contribute to ATP formation. This is a clas-
sical interpretation, but an incorrect one. The complexes are not physical sites of ATP forma-
tion but merely separate contributors to the proton gradient. To appreciate the central role
of the proton gradient, we will first investigate the complexes in some detail, the explanation
for how the proton gradient is utilized, and the phenomenon of uncoupling, strong evidence
of the involvement of the proton gradient in energy production.
11.7 MECHANISMS OF THE MITOCHONDRIAL

MEMBRANE PROTEIN COMPLEXES
A full understanding of the proton and electron flows across the mitochondrial membrane
requires a more detailed description of the five complexes. In this section, we will see how the
244 11.7 Mechanisms of the Mitochondrial Membrane Protein Complexes
first four complexes are responsible for electron and proton flow to create a proton gradient.
Complex V is the conduit for proton re-entry and ATP synthesis.
11.7.1 COMPLEX I: PROTON PUMP
The first complex is also known as NADH-UQ reductase. It accepts electrons and pro-
tons in the form of a hydride ion from NADH, donates electrons to the mobile carrier
UQ, and pumps protons across the membrane in the process. Mammalian NADH-UQ
reductase is composed of a large number of subunits – 45 protein chains – and contains
the bound flavin FMN, in addition to several Fe–S proteins. Structural studies so far have
provided only an outline of how Complex I functions, but its operation is well under-
stood. Complex I can be divided into three general portions (Figure 11.9): the N-module,
the input domain that directly reacts with NADH; the P-module, through which protons
are transported; and the Q-module, which reacts with UQ. The N-module contains the
bound FMN along with several Fe–S proteins, so that electron flow is from NADH to
FMN to Fe–S. Here, the transducer role of the flavin is apparent, as FMN accepts a pair
of electrons in the form of a hydride, but donates them one at a time to an Fe–S cluster.
Protons take a separate route, passed to side chains of the protein subunits until they are
finally released through the P-module. The nature of “proton pumping” can be visualized
as a conformational change that situates a proton on the matrix face first and then the
cytosolic side. Figure 11.10 shows that the process is analogous to the action of the glucose
transporter. When no high energy electrons pass through the complexes, a proton bind-
ing site faces the matrix. As electrons flow, a conformational change flips the structure
and the proton binding site faces the cytosol. Reaction at the Q-module (Figure 11.9) takes
electrons from the Fe-S clusters and protons from the matrix space to produce UQH 2 , the
reduced form. Note that both UQ and UQH 2 are mobile entirely within the hydrophobic
portion of the membrane.
FIGURE 11.9 Complex I: NADH-Q reductase. There are three modules to this complex, called
N, P, and Q. The N-module is the site of electron entry, where NADH reduces the flavin FMN. The
P-module is where proton pumping occurs. The Q module is where electrons, along with pro-
tons from the matrix, attach to UQ. Most of the electron transport involves Fe-S proteins, some
of which are indicated.
FIGURE 11.10 Proton pumping analogous to glucose transport. Like the glucose transporters,
proton pumping can be viewed as binding to a protein that faces one water space or the other
in a membrane. In electron transport, the energy driving the conformational change is from
electron transport through the protein, which alters the local electrical potential.
11.7.2 COMPLEX II: SUCCINATE DEHYDROGENASE
Complex II is synonymous with succinate dehydrogenase, also a step in the Krebs

cycle. Thus, this reaction sequence plays a dual role in central metabolic pathways. The
sequence of reactions is analogous to Complex I: a hydride is transferred to a flavin
cofactor, and the electrons are then passed to a series of Fe-S complexes and finally to
UQ. In the case of succinate dehydrogenase, the hydride comes from succinate, the fla-
vin is FAD, and no protons are translocated in the overall process. Electrons from both
Complexes I and II converge in UQH 2 formation, as do other complexes within the mito-
chondrial membrane. These include glycerol phosphate oxidase, which we will examine
at the end of this chapter, and electron transferring flavoprotein, an enzyme of fatty acid
oxidation (Chapter 14). All of these reaction sequences produce UQH 2 , which is oxidized
exclusively by complex III.
11.7.3 COMPLEX III: LOOP MECHANISM
Complex III is also known as UQ-cytochrome c reductase, because it takes electrons from
UQH2 and reduces cytochrome c. Unlike the other complexes of the respiratory chain, elec-
tron flow is partially cyclic in complex III; the mechanism is referred to as a loop mechanism
or the Q-cycle. Similar to the Krebs Cycle, the pathway of electron flows in the Q-cycle is an
example of repetition–variation. As illustrated in Figure 11.11, more than one traversal of the
cycle is necessary to balance the overall reaction. The membrane faces are labelled N-side for
the matrix (as it becomes negatively charged as protons move across the membrane to the
cytosol), and the P side (positive) for the cytosolic face.
Starting with the reduced form, UQH2 from Complex I or II, for example, let us follow
the first coenzyme, labeled (1) in Figure 11.11. At the P side membrane face, the quinone dis-
sociates both protons, and donates an electron to an Fe/S protein, forming the radical anion
UQ●−. From the Fe–S protein, the electron is donated to cytochrome c1 within the complex
and next to the mobile cofactor cytochrome c. These events are depicted in the inset to
Figure 11.11. UQ●− is an amphiphile; it is localized to the P side membrane as its charged
portion faces the cytosolic water space, and the remainder of the molecule remains embed-
ded in the membrane. The localized negative charge of the amphiphile UQ●− is the reason
the molecule is fixed to the membrane surface. In the next step, an electron is donated to
a b cytochrome labeled bL , and the fully oxidized form of ubiquinone, UQ results. Like the
fully reduced form, UQ is mobile within the lipid portion of the membrane, and can dif-
fuse to the N side. Meanwhile, the reduced cytochrome bL passes an electron to a second b
cytochrome, labeled bH, which subsequently reacts with UQ at the N side to form UQ●−. The
radical ion at the N side forms a distinct pool from the UQ●− at the P side, a point proven
FIGURE 11.11 The Q cycle. Both ubiquinone cofactors and electrons cycle through the mem-
brane. The pathway can be understood by breaking it down into two discrete steps that can join
up in the steady-state. In pathway (1), drawn in black, UQH2 dissociates, releasing two protons to
the cytosol and one electron to an Fe-S protein (see inset for detail). The electron subsequently
is passed to cytochromes c1 and c. The form of ubiquinone remaining is the intermediate single-
electron reduced UQ●− at the cytosolic membrane leaflet (P side). Next, electrons from UQ●− are
passed to cytochrome bL, and the quinone is in its fully oxidized form, UQ, which can diffuse
across the membrane to the N side to UQ●−. Electron flow continues as the reduced bL passes
its electron to bH, and then reduces the UQ that has arrived at the N side. At this point, we leave
pathway (1). Pathway (2) is indicated in red. The same initial steps of pathway (1) convert the sec-
ond molecule of UQH2 to UQ●−. From there, the pathway proceeds in the same way as pathway
(1) to produce UQ and reduced bH. The UQ is abandoned for this leg, as the electron from bH is
donated to UQ●−, and protons removed from the matrix (that is, the N side) to form UQH2. The
overall cycle can be completed only in the steady-state.
by experiments using electron spin resonance showing two distinct spectra in respiring
mitochondria.
At this point, the pathway cannot continue without another electron to fully reduce the
quinone. This second electron arises from the second molecule of UQH2, illustrated as path-
way (2) and colored in orange in Figure 11.11. This second UQH2 undergoes the same reac-
tions to form UQ●− at the P side, and then UQ as it passes electrons to bL. However, for this
half of the mechanism, we must orphan the UQ and continue with electron flow from bL
to bH. The electron from bH can be seen to complete the reduction of N-side UQ●−, taking
protons from the mitochondrial matrix, and forming UQH2. The diffusion of UQH2 to the
P-side completes the cycle.
While the description of the Q cycle is necessarily disjoint, in the steady-state, there is a
continuous flow of electrons both through the membrane – that is at the P side, from UQH2
to cytochrome c – and traversing the membrane through the two b cytochromes. The latter
is utilized only to complete the loop. The operation of this loop removes protons from the
mitochondrial matrix, carries them as the hydrogens of UQH2, and deposits them to the
cytosol as the quinone is oxidized at the P side.
We should appreciate that not only are there two signatures for UQ●−, but also separate
signatures for cytochromes bL and bH. In the case of the quinone, there are two distinct
spin states (electron paramagnetic resonance detects unpaired electrons). In the case of
cytochrome b, there are two different light absorbance wavelengths. In each case, the rea-
son for the distinction is the different electrical field across the membrane, established by
the movement of protons, making the N side relatively negative and the P side relatively
positive.
11.7.4 COMPLEX IV: PUMP AND ANNIHILATION
The final complex of electron transport is also known as cytochrome c oxidase. Like
Complex I, the pumping of protons is tied to conformational changes in protein subunits
as high-energy electrons are passed through the complex. Figure 11.12 shows the path of
electron flow. After accepting electrons from Complex III, cytochrome c slides along the
cytosolic face of the membrane and donates electrons to CuA, a dinuclear copper center,
which contains two copper ions joined by sulfur atoms (Figure 11.13), a structure analogous
to the iron-sulfur complexes. The path of electron flow continues through cytochrome a,
then cytochrome a3, which is associated with another copper center, CuB, in which the ion
is chelated to three histidine groups (Figure 11.13). Overall, four electrons flow through the
complex, and two water molecules are formed from O2. The water synthesis step – the ter-
minal stage of electron transport in the chain – involves oxygen bound alternatively between
CuB and cytochrome a3 in an interior portion of the protein complex. Note that the CuB ions
as well as cytochrome a3 have dual roles: binding oxygen (like the non-redox-active hemo-
globin) and undergoing redox state transitions, as electrons are moved through complex IV.
Complex IV has two mechanisms for creating the proton gradient. One of these is a pump
mechanism, similar to that involved in Complex I (Figure 11.9). As electrons move between
cytochromes a and a3, the altered electrical potential induces a conformational change in a
protein, flipping a proton-bound face from matrix to cytosol. The second mechanism is an
annihilation of protons, specifically from the matrix side. These protons go into the synthe-
sis of water. Because a loss of protons from the matrix is equivalent to a gain of protons to the
cytosol, annihilation contributes to the proton gradient.
11.7.5 COMPLEX V: ATP SYNTHESIS
The movement of protons in the direction of cytosol (P side) to matrix (N side) is catalyzed by
complex V, which has a tunnel-like passageway for protons and an active site for the synthesis
FIGURE 11.12 Complex IV: Cytochrome c oxidase. Electrons from cytochrome c are donated
to CuA, a cofactor pictured in Figure 11.13. Subsequently, electrons are passed to cytochrome
a. As electrons move next to cytochrome a3, a conformational change drives proton pumping,
in a way similar to Complex I. Electrons are passed to the cofactor CuB (a structure also shown
in Figure 11.13) and lastly to water. Protons consumed in water formation also contribute to the
proton gradient, a mechanism called annihilation.
FIGURE 11.13 Copper centers. Structures of copper centers participating in electron transfer
in Complex IV.
of ATP from ADP and Pi. The complex is also known as the F1FoATP synthase. One por-
tion of the complex – called F1 (factor one) – binds adenine nucleotides and is responsible
for ATP synthesis. The other – called Fo (oligomycin sensitive factor) – conducts protons
through the membrane. Early micrographs of the complex showed that Fo is anchored to
the membrane, and F1 protrudes into the matrix like a lollypop. The determination of the
mechanism for complex V was, in large part, the work of Paul Boyer. A series of enigmatic
findings had to be resolved. First, the binding of ATP to the complex was found to display
negative cooperativity.
Thus, for the three ATP binding sites, the first has a dissociation constant of less than
1 nM; the second, 1 μM; and the third, 30 μM. In contrast to binding, enzymatic activ-
ity exhibited just the opposite behavior – namely, as more ATP molecules were bound, the
activity increased. This situation could be explained if the limiting step in the overall reac-
tion is the release of the nucleotide. Somehow, the enzyme complex would have to undergo
a conformational change that would release ATP, and this had to be related to the entry of
protons. Boyer proposed that a protein segment of the complex might move from one of
the three ATP binding sites to the other, in concert with the proton entry to drive the con-
formational change that elicits ATP release. This mechanism was named binding change.
Further development of this mechanism used an analogy from the operation of a motor
(Figure 11.14). The two parts are the moving rotor and the stationary stator. In this depiction,
the movement is driven entirely by magnetism; repulsion of like magnetic poles causes the
rotor to spin with respect to the stator. Real motors use electromagnetic induction, but the
principle is the same. The detailed subunit structure of complex V is shown in Figure 11.15.
Structures that correspond to the stator are shaded in red, while those corresponding to the
rotor are shaded in green.
FIGURE 11.14 Essence of a motor. The two essential components of a motor are the stator
and the rotor. In this simplified diagram, the repulsion of fixed magnets is the driving force for
rotation. In a real motor, electromagnetic induction is used to drive the rotor.
FIGURE 11.15 Complex V: ATP synthase as a molecular motor. The components ATP synthase
(a) are colored to correspond to the motor of Figure 11.14: parts of the stator are in red and parts
of the rotor in green. Various protein subunits are labeled with both Greek (exterior portions) and
Roman letters (largely membrane-embedded portions) by convention. The rotor tip is hidden by
the three pairs of αβ proteins of the stator. The diagram is sketched to be partly visible. A top view
(b) of the edge of the rotor (γ) rotating and interacting intermittently with the ATP-binding por-
tion of the stator (β) shows the action of the enzyme: as each stationary unit interacts with the
rotor, ATP dissociates. Dissociation of ATP is the rate-limiting step of the overall reaction. In the
side view (c), it is apparent that the stator and rotor closely interact in the membrane segment,
and, as the protons pass between these, it moves the rotor.
250 11.8 Proton-Motive Force
11.7.5.1 THE STATOR
Three αβ dimers are arranged as a cylinder, attached to a stalk made of b–subunits. The
dimers are attached, in turn, to a membrane-embedded a–subunit. The cylinder of αβ sub-
units comprises most of the knob structure of the previously mentioned F1 that appears in
electron micrographs. Adenine nucleotides bind to the β subunit of the αβ dimers.
11.7.5.2 THE ROTOR
A ring of membrane-bound c subunits is attached to a stalk containing ε and γ subunits.

The γ subunit can contact αβ dimers sequentially, which changes the conformation of the
β subunits. This conformational change is key to the reaction as indicated in Figure 11.5b.
Each of the three stages of ATP synthesis is represented by one of the three states of the β
subunit: the binding of ADP and Pi to the subunit; bond formation to synthesize ATP; and
the release of ATP. The moving rotor supplies the conformational change to drive each
of these states in turn. Proton entry occurs at the interface between the stator and rotor,
specifically at the junction between the a and c subunits, as shown in Figure 11.15c. As the
protons move in, the c subunits rotate, as does the connected γ-subunit, which spins among
the three αβ dimers. In this way, the entry of protons is indirectly coupled to ATP synthesis.
Complex V can be experimentally studied in the reverse direction in vitro; it is the only
direction that can be observed unless the mitochondria are intact. The in vitro activity is
a splitting of experimentally added ATP into ADP and Pi, spinning the rotor in the reverse
direction to synthesis.
11.8 PROTON-MOTIVE FORCE
The detailed description of the complexes just presented provides insight into electron and
proton pathways of the inner membrane (Complexes I, III, and IV), and how the proton gra-
dient thus created can be used to synthesize ATP (Complex V). In the steady-state, a gradient
of protons always exists across the inner membrane, known as the proton-motive force.
We have already observed the analogy between the electromotive force of a battery and
the proton gradient across the mitochondria (Figures 11.5 and 11.6). The proton gradient
can be considered to have two components: an electrical one, or membrane potential, and a
chemical one, a pH gradient. The proton-motive force is thus the electrochemical potential
for a proton, the gradient created by the respiratory chain, and utilized for ATP formation.
The proton-motive force is symbolized as Δp, and can be written as a Nernst equation (intro-
duced in Chapter 8):
(11.3) Dp = DY - 60 * DpH
Where ΔΨ is the electrical potential across the membrane (negative to the inside), the −60
incorporates the term RT/F at room temperature, and the logarithmic concentration gradi-
ent is expressed as ΔpH. All terms are measured in millivolt units.
Equation 11.3 emphasizes the two contributions to the gradient: the electrical and chemi-
cal differences. In mitochondria, a typical value for Δp is −200 mV, of which the electri-
cal contribution (ΔΨ) accounts for about 80%. A common misconception is to consider the
proton gradient a “pH gradient”. Actually, the chemical contribution is quantitatively more
minor.
11.8.1 ELECTRIC CIRCUIT ANALOGY TO THE PROTON GRADIENT
Just as we considered the protonmotive force as analogous to an electrochemical cell, we can

imagine the operation of the mitochondria in direct analogy to an electric circuit. The circuit
containing a battery and a lamp is drawn with an open switch in Figure 11.16a and a closed
FIGURE 11.16 Electrical analogy to ATP synthesis. The electric circuit in (a) is a battery con-
nected to a lamp with an open switch. When the switch is closed, the lamp glows. Notice the
voltage across the battery in the open circuit is 3 V, but drops to 2.8 V when the switch is closed.
If the switch is opened again, the voltage will return to 3 V. The analogous changes in mitochon-
drial voltage (Δp) are shown in (c) and (d). The situation in (c) is like the open switch of (a). In
(c), however, it is the proton that is the mobile circuit element, and rather than an open switch,
oligomycin is present to block the completion of the circuit. In the complete circuit (d), protons
cycle across the membrane, and ATP is formed. Notice that the voltage drops when the circuit is
complete, just like that of the electrical circuit.
switch in Figure 11.16b. Also indicated are the voltages across the battery (and of course the
lamp): 3 V for the open circuit and 2.7 V for the closed circuit. While a simple device, we note
that the dissipation of energy (light and heat in this case) at the lamp has decreased the volt-
age across the battery. The voltage drop reflects the conversion of potential to kinetic energy.
If the switch is opened again, the battery voltage will once again read close to 3 V.
Now consider the mitochondrial model of Figure 11.16c. In this case, the protons are
moving through the electron transport chain symbolized as a flat oval. They are blocked
from re-entry by the presence of oligomycin. Oligomycin prevents proton entry through the
Fo component of ATP synthase; the “o” stands for oligomycin. The situation of Figure 11.16c
is analogous to the incomplete circuit of Figure 11.16a, and the voltage (Δp) across the mem-
brane is shown to be −200 mV. In the absence of oligomycin, protons can enter the mitochon-
drial matrix, and the proton circuit is complete, as shown in Figure 11.16d. Now the proton
circuit is analogous to the complete electrical circuit of Figure 11.16b. The ATP synthase uti-
lizes the proton gradient produced by the respiratory chain. Notice the Δp in Figure 11.16d
has dropped somewhat. Just like the lamp of Figure 11.16b, the ATP synthase is a “load” for
the circuit and decreases the potential.
11.8.2 UTILIZATION OF ΔP BEYOND ATP SYNTHESIS
There are other uses for the energy created in the form of Δp by mitochondria beyond ATP
synthesis. One of these is a reaction known as the energy-linked transhydrogenase, a
membrane-bound enzyme that converts NADH to NADPH by the reaction:
(11.4) NADH + NADP+ + éëH + ùû ® NAD+ + NADPH + éëH + ùû

cyto mito
252 11.9 Mitochondrial Membrane Transport
The enzyme is embedded in the inner membrane and provides an input pathway for protons
and an active site inside the mitochondrial matrix where it exchanges the hydride of NADH
for that of NADPH. The latter nucleotide is used in several reactions within mitochondria,
including glutathione reduction to reduce reactive oxygen species. Reaction 11.4 is metaboli-
cally irreversible so long as the mitochondria maintain a Δp – that is, so long as it is viable.
Most reactions that utilize the Δp involve transport of materials across the inner mem-
brane; these are described in the following section. Finally, an alternative use of the pro-
ton gradient is simply a short-circuit, a situation known as uncoupling, which is described
subsequently.
11.9 MITOCHONDRIAL MEMBRANE TRANSPORT
Several embedded proteins accomplish selective exchange across the mitochondrial mem-
brane. Most utilize the Δp of the mitochondria, either the electrical portion or the chemical
portion of the proton gradient. Transport proteins that catalyze exchange with a simulta-
neous dissipation of a portion of the ΔΨ are called electrogenic. All known electrogenic
exchanges are metabolically irreversible. The majority of exchangers either utilize the chemi-
cal gradient of protons (i.e., ΔpH) or else do not alter the Δp. These exchangers are at near-
equilibrium and can operate in the opposite direction.
11.9.1 ADENINE NUCLEOTIDE TRANSLOCASE
As most of the ATP synthesized by mitochondria is needed in the cytosol, it must be

exported. The adenine nucleotide translocase takes fully charged ATP4− from the matrix side
and ADP3− from the cytosolic side (Figure 11.17a). The result is a net movement of negative
charge out of the mitochondria, meaning that the membrane potential drives the transport
in one direction. The translocase is thus metabolically irreversible. The exchange reaction is
the only metabolic step involving ATP that does not require chelation with Mg2+. Because the
ATP/ADP exchange is driven by the ΔΨ, it is an electrogenic transporter.
11.9.2 PHOSPHATE EXCHANGE
Inorganic phosphate can cross the mitochondrial membrane through more than one trans-
porter. Two are shown as Figures 11.17b and 11.17c. The Pi−/H+ cotransporter directly draws
on the ΔpH portion of the mitochondrial proton-motive force. This exchanger acts at near-
equilibrium and can operate in the opposite direction. There is also is a near-equilibrium
exchange of Pi for malate. The compounds are both exchanged in the −1 charge form. For
both Pi exchangers, there is no alteration in membrane potential (i.e., they are electroneutral,
not electrogenic).
11.9.3 OTHER TRANSPORT PROTEINS
Other mitochondrial membrane transporters can also be classified as either electroneutral or

electrogenic (Figure 11.18). For example, the α-ketoglutarate/malate exchanger is an electro-
neutral, near-equilibrium transport. The glutamate/aspartate exchanger and the Ca2+ uptake
uniport are both electrogenic, metabolically irreversible steps. Like the adenine nucleotide
transporters, these draw upon the membrane potential to drive glutamate0 into the matrix
and aspartate−1 out to the cytosol, making the matrix more positive. Similarly, Ca 2+ uptake
into the matrix is electrogenic, bringing with it two uncompensated charges. While these
steps cannot be reversed, they are not likely to be regulatory steps either, because they
respond only to concentration changes.
FIGURE 11.17 Mitochondrial transporters. Three transport systems of the mitochondria are
illustrated. (a) ATP/ADP exchange is electrogenic, driven by the membrane potential. (b) Pi−/H+
exchange is driven by the concentration differences in H+ ion (ΔpH). (c) Malate/Pi exchange is
driven exclusively by changes in concentrations of the components.
11.9.4 COUPLING OF OXIDATION AND PHOSPHORYLATION
We have already considered the F1FoATP synthase, as a key part of oxidative phosphorylation.
In one sense, it is also a transport protein. As shown in Figure 11.17, it facilitates transport
of a proton into the mitochondria. Unlike the other transporters, it draws on both aspects of
the Δp since it is the proton itself that is transported. Also distinguishing Complex V is the
fact that it catalyzes ATP synthesis as protons are imported.
We can consider the proton gradient produced by the respiratory chain as a driving force
for three events needed to form cytosolic ATP: (i) the conversion of ADP and Pi to ATP (ATP
synthase), (ii) the uptake of Pi and (iii) the exchange of cytosolic ADP for mitochondria ATP.
These events can only be completed as a pathway in the steady-state. For example, there
would be no ATP to exchange with the first molecule of ADP until a separate ATP synthesis
event occurs.
The linkage of ADP phosphorylation with Δp-forming electron transport is known
as the coupling of oxidation to phosphorylation; hence the key energy-forming opera-
tion of mitochondria is known as oxidative phosphorylation. The idea of coupling oxi-
dation with phosphorylation predates the chemiosmotic hypothesis that explains how
mitochondria make ATP. The early experiments were direct and compelling: when mito-
chondria were provided with substrates that could supply NADH and with Pi, the addition
of ADP caused a large increase in oxygen consumption from the media. Of course, the
254 11.10 Uncoupling
FIGURE 11.18 Further mitochondrial transporters. Three more transporters within the mito-
chondrial inner membrane are illustrated: α–ketoglutarate−/malate− exchange responds only
to concentration changes; glutamate0/aspartate− exchange, on the other hand, is electrogenic,
driven by membrane potential; Ca2+ uptake is a uniport, which is also electrogenic and driven by
the membrane potential.
chemiosmotic hypothesis explains this and also enables an understanding of the mecha-
nisms of the proton-conducting complexes. As we have already seen, the movements of
electrons and protons through the respiratory chain are interlinked. The coupling of oxi-
dation with phosphorylation, on the other hand, is indirect, through the proton gradient.
This is dramatically illustrated by what happens when this coupling link is broken – a
situation called uncoupling.
11.10 UNCOUPLING
The link between oxidation and phosphorylation can be broken in a way that allows respi-
ration to continue without the synthesis of ATP. Historically, compounds that caused this
phenomenon of uncoupling were difficult to categorize because there were many of them,
and they varied in structure. Because earlier hypotheses about mitochondrial energy genera-
tion postulated a chemical intermediate of high energy, it was hard to see how a large array
of different compounds could specifically interact with it.
The chemiosmotic hypothesis resolved the mystery. If the intermediate is a proton gra-
dient, and protons normally enter mitochondria via the F1FoATP synthase, an alternative
pathway that bypasses Complex V but still allows the proton entry into the mitochondrial
matrix would act as an uncoupler. Certain weak acids can cause uncoupling. A mechanism
for the uncoupling of a weak acid is diagrammed in Figure 11.19a. In its protonated form, HA
crosses the membrane – without a transporter – and dissociates in the matrix into H+ and
A−. The A− returns to the exterior, where it can combine with a proton that has been driven
out of the mitochondria by the electron transport chain, form HA, and completes the cycle
by entering the matrix again.
Only certain types of weak acid can cross the membrane both in the HA and the A− forms.
There are two requirements. First, both forms must be small lipophilic molecules so that they
FIGURE 11.19 Uncoupling. The general process is illustrated in (a). The proton produced
by the respiratory chain reacts with an anion symbolized by A− to form the weak acid, HA. HA
crosses the membrane without a carrier, dissociates upon entry to the mitochondrial matrix, and
returns the proton back to the matrix. The anion, A−, can also cross the membrane, driven by the
electrical gradient (i.e., the membrane potential). (b) The structures of the classical inhibitor DNP
(dinitrophenol) in HA and A- forms. (c) The structure of CCCP (carbonyl cyanide m-chlorophenyl
hydrazone), a more potent inhibitor that is commonly used experimentally.
can traverse the membrane in the absence of a transporter. Second, the charge of the A− form
must be delocalized so that it too can pass through the membrane. If the charge is localized
to a portion of a molecule, it would be impermeant to a membrane.
One common molecule that satisfies these criteria is dinitrophenol (DNP) (Figure 11.19b).
The aromatic ring in the charged form distributes the excess electron throughout the ring as
well as through the nitro groups.
It is also possible to have uncouplers that are weak bases (conjugate acids), so that they
enter the matrix in their positive form when protonated and leave uncharged. Currently,
popular uncouplers used experimentally are of precisely this type, such as the compound
abbreviated CCCP (Figure 11.19c). A large number of drugs are hydrophobic because this
helps them enter cells for therapeutic purposes. Many of these are also weak acids (or con-
jugate acids), so they may act as uncouplers. It is standard practice to test candidate drugs
for their potential as uncouplers as part of a toxicology screen. The classical uncoupler, dini-
trophenol, was itself marketed in the 1930s as a diet drug before its full toxic qualities were
realized (Box 11.2).
256 11.11 Superoxide Formation by Mitochondria
Box 11.2 The Once and Future Uncoupling Diet
During World War I, dinitrophenol (DNP) was used as an explosive. This action, like the
structurally similar trinitrotoluene (TNT)
OH OH
NO2 O 2N NO2
NO2 NO2
DNP TNT
is the result of a rapid release of its components into gases; in particular the nitro groups
are particularly unstable. A group of French munitions workers exposed to DNP expe-
rienced weight loss. A pharmacologist studied low doses of DNP as a diet drug in the
1930s and marketed it as “Formula 281” (“too weighty one”). The product was extremely
popular. However, even slight excesses of DNP led to cataracts, blindness, and death.
There can be little doubt about its effectiveness: by allowing respiration but attenuating
ATP formation, uncouplers such as DNP permit overeating without weight gain. Despite
the toxicology of uncouplers, DNP has re-emerged as a diet pill thanks to the Internet and
counterculture viewpoints.
There is a possible future for the “uncoupler diet”. As described in the text, the uncou-
pling protein found in brown adipose tissue functions as an endogenous uncoupler.
Thus, there is the possibility that genetic manipulation of brown fat may become a will-
power-free diet. Until then, we are left with the only diet that actually works: eat less,
exercise more.
11.10.1 PHYSIOLOGICAL UNCOUPLING: BROWN FAT
In certain modified fat cells, the mitochondria contain a protein known as UNC or uncou-
pling protein. There are several isoforms, but UNC1 is the best studied. UNC1 is known to
conduct protons back into the mitochondrial matrix of brown fat, bypassing ATP synthesis.
Like exogenous small molecule uncouplers, activation of UNC1 causes increased cellular
respiration, as well as an increased temperature, as the energy is dissipated as heat rather
than in the synthesis of ATP. Hibernating bears activate their brown fat as part of the return
to their active state. Generally, brown fat is scarce in humans apart from a small amount in
newborns.
In the last several years, the presence of brown fat and conditions that lead to its activa-
tion in adult humans (such as exercise and certain hormones) have been identified. Brown fat
can arise by a transformation of ordinary white fat. While use of a small molecule uncoupler
like DNP is a dangerous way to diet, an elevation of brown fat would be an ideal means of
overcoming obesity.
11.11 SUPEROXIDE FORMATION BY MITOCHONDRIA
Oxygen can abstract electrons from intermediate sites in the electron transport chain to pro-
duce superoxide (O2−), an oxygen molecule with an extra electron. Superoxide is extremely
reactive with cellular components, particularly some lipids and proteins, and can precipitate
cell death.
For many years, it was believed that oxidative damage was associated with premature
aging. However, extensive studies performed in 2009 found no correlation between oxida-
tive stress and longevity across several species. This is likely due to cellular systems that
protect against oxidative damage by the inactivation of superoxide and related species. First,
superoxide dismutase catalyzes the formation of the more stable hydrogen peroxide (H2O2).
Subsequently, peroxidase catalyzes the conversion of hydrogen peroxide to water. Similar
inactivation enzymes also exist within the mitochondrial matrix space.
There are two sites in the respiratory chain where superoxide can be formed, both of
which are inferred from the presence of cytosolic and mitochondrial detoxification systems.
Matrix superoxide can be formed at Complex I; however, like the mechanism of Complex I
itself, details of this process remain unclear. Cytosolic superoxide is formed from the reac-
tion of the cytosolic-facing radical ion of ubiquinone:

(11.5) UQ ·- + O2 ® UQ + O2·-
In both cases, electrons drawn into superoxide formation result in fewer electrons flowing
through the electron transport chain. It turns out, however, that only a small percentage of
electrons end up in superoxide.
11.12 CONTROL OF MITOCHONDRIA
The primary function of mitochondria is to produce ATP for use by cytosolic reactions. Most
of that ATP is converted to ADP. It is fitting, then, that the concentration of ADP is the prin-
cipal means of controlling further ATP formation by mitochondria.
As ADP enters the mitochondria, it is available to the F1Fo ATP synthase, which admits
protons into the matrix as it synthesizes ATP. Proton entry completes the circuit as described
by analogy to electrical circuitry in Figure 11.16. We observed that the presence of the inhibi-
tor oligomycin could stop the proton circuit. A different way of altering the circuit flow is by
the availability of ADP itself. Suppose no ADP was available. In that case, the Δp would be
at its high point (−200 mV), the proton circuit would stop, and no ATP would form. As more
ADP is available, the respiratory chain responds with increasing amounts of proton flow, elec-
tron flow, and ATP formation. Control of oxidative phosphorylation by ADP is regulation at
a fundamental level, establishing a connection between energy demand, as reflected in cyto-
solic ADP concentration, and mitochondrial energy supply, as reflected in ATP formation.
The total cytosolic ADP concentration of cells exceeds the free solution concentration by
an order of magnitude, as most ADP exists bound to cytosolic proteins. It is only the free
[ADP] that is significant for the regulation of mitochondrial energy production. Because of
the discrepancy between free and total [ADP], measurements of the total concentration of
this nucleotide are unrelated to cellular energy production.
Control by ADP concentration assumes that the supply of electrons, largely from NADH,
are not themselves limiting. This could be the case if there is insufficient nutrient supply,
from glucose or fatty acids, to maintain cell function. This is not, however, a physiological
state but rather a condition of starvation that could limit mitochondrial function. At the
other extreme, overfeeding does not in itself control mitochondrial energy production. In
that case, excess substrates are mainly converted into lipids for storage (Chapter 14)
The adenine nucleotide translocase, which exchanges cytosolic ADP for mitochondrial
matrix ATP, may be thought to also limit oxidative phosphorylation, as this protein is not
in excess and is metabolically irreversible. However, the exchanger responds directly to the
concentration of cytosolic ADP. While metabolic irreversibility is a condition for a step to be
considered as a control point, it does not mean that it must be. Strictly speaking, metabolic
irreversibility means only that the reaction cannot be considered to act in reverse under cel-
lular conditions.
Another transporter that utilizes membrane potential is the mitochondrial Ca2+ uni-
porter (introduced in Section 11.9.3 Other Transport Proteins). Calcium uptake into the
258 11.13 How Mitochondria Can Utilize Cytosolic NADH
mitochondria driven by the uniporter depends upon changes in cytosolic [Ca2+]. In turn, the
cytosolic Ca2+ is dependent upon the amount released from the endoplasmic reticulum in
response to external cellular regulation, such as electrical depolarization or hormonal acti-
vation. Elevation of mitochondrial [Ca2+] activates the Krebs cycle and pyruvate oxidation.
While very high rates of mitochondrial Ca2+ accumulation could decrease the membrane
potential enough to limit ADP uptake, this is a pathological rather than a physiological con-
dition. A massive increase in cytosolic [Ca2+] is known to elicit cell death.
11.13 HOW MITOCHONDRIA CAN UTILIZE CYTOSOLIC NADH
In the complete oxidation of glucose to CO2, we calculated the energy balance (Section 10.5)
as 38 ATP per glucose molecule. The rationale for assuming three ATP per NADH and two
ATP per UQH2 requires an estimation of the extent of ATP formation based on the proton
gradient formed. The measurement of proton numbers formed in the chain varies somewhat
between laboratories, but all are slightly less than 3 for NADH and slightly less than 2 for
QH2. The whole number values are sufficient for comparative purposes.
There is a separate issue that we can now address: if the glycolytic pathway produces
pyruvate that is fully oxidized by mitochondria, how is the cytosolic NADH reoxidized?
Since net lactate formation is bypassed in aerobic metabolism, the NADH produced at
glyceraldehyde P DH in the cytosol must be oxidized by mitochondria. However, there is no
mitochondrial transporter for NADH (or any nicotinamide nucleotide). Instead, there are
two pathways that indirectly oxidize cytosolic NADH by transferring just its electrons or
reducing equivalents. These are examples of shuttle pathways.
11.13.1 GLYCEROL PHOSPHATE SHUTTLE
The simpler of the shuttles illustrates the general pattern: two redox reactions utilize the
same pathway intermediates in two metabolic spaces – except in opposite directions. For the
glycerol phosphate shuttle, the cytosolic enzyme is glycerol phosphate dehydrogenase. The
enzyme catalyzes the near-equilibrium reaction:

(11.6) DHAP + NADH ® Glycerol-P + NAD+
The second reaction, which is metabolically irreversible, is catalyzed by glycerol phosphate

oxidase, an enzyme complex located in the inner membrane:

(11.7) Glycerol-P + UQ ® DHAP + UQH 2
The reactive site for glycerol-P is on the cytosolic face of the membrane.
The operation of both of the reactions creates a short cyclic pathway, which serves to move
reducing equivalents from cytosolic NADH to the membrane resident mobile carrier, UQH 2.
The sequence is illustrated in Figure 11.20, which also shows the key components of the glyc-
erol phosphate oxidase complex. The latter is strikingly similar to succinate dehydrogenase.
Like Complex II, glycerol phosphate oxidase does not contribute to the proton gradient.
In essence, the oxidation of cytosolic NADH through the glycerol phosphate shuttle
bypasses Complex I and the protons it pumps. Only about 2/3 of the energy of mitochondria
can be realized by introducing electrons as UQH2 compared to NADH. A second shuttle,
called the malate/aspartate shuttle, while more elaborate, provides a greater energy yield.
11.13.2 MALATE/ASPARTATE SHUTTLE
The malate/aspartate shuttle shares the same features we have just noted in the glycerol
phosphate shuttle for importing reducing equivalents: an overall cyclic pathway with at least
FIGURE 11.20 The glycerol phosphate shuttle. Reducing equivalents from NADH are brought
into the mitochondria by the operation of two enzymes. The cytosolic GOLPDH (glycerol phos-
phate DH) reduces DHAP (dihydroxyacetone P) to form GOLP (glycerol P). Subsequently, the
GOLP reacts with the GOLP oxidase complex in the mitochondrial membrane at the cytosolic
face, and electrons are shuttled through the complex to FAD, Fe/S proteins, and then to the
mobile cofactor UQ.
one metabolically irreversible step to drive the flow in just one direction, with duplicate reac-
tions in separate metabolic spaces, running in opposite directions.
The malate/aspartate shuttle uses two sets of enzymes, rather than one, because the inter-
mediate oxaloacetate, like NADH, has no transporter in the mitochondrial inner membrane.
The pathway is illustrated in Figure 11.21. Note that it involves two isozymes of malate DH:
the cytosolic enzyme, which oxidizes NADH, and the mitochondrial enzyme, which reduces
NAD+. Malate is imported to the mitochondria in exchange with α-ketoglutarate (as in
Section 11.9.3 Other Transport Proteins). Because oxaloacetate itself cannot be transported
out of the mitochondria, it is converted to aspartate through the aspartate aminotransferase
reaction:

(11.8) Oxaloacetate + Glutamate ® Aspartate + a-Ketoglutarate
Aspartate can exit the mitochondria in exchange for glutamate. As discussed in Section
11.9.3, this transport step is electrogenic and hence metabolically irreversible. In fact, it is
the only step of the entire shuttle that is metabolically irreversible, and it accounts for the
unidirectionality of the shuttle. A cytosolic isozyme of aspartate aminotransferase catalyzes
the reconversion of aspartate to oxaloacetate.
The malate/aspartate shuttle is more elaborate than the glycerol phosphate shuttle. For
example, rather than just two reactions, the malate/aspartate shuttle requires six. The cycle
is neatly balanced as glutamate and α-ketoglutarate needed at the transamination steps are
transported in the correct directions in the operation of the pathway. Despite the apparent
complexity, this is by far the more common route for the delivery of reducing equivalents
into the mitochondria.
We will see some of the same reactions of the malate/aspartate shuttle utilized in other
pathways (and shuttles applied more generally), in the gluconeogenic and lipid pathways, and
in nitrogen metabolism. However, before we study additional pathway connections, we will
260 Summary
FIGURE 11.21 The malate-aspartate shuttle. Reducing equivalents from NADH are trans-
ferred to malate via the malate DH (MDH) reaction. Once inside the mitochondria, malate
reacts with a separate MDH, producing mitochondrial NADH. Two aspartate aminotransferase
enzymes (AAT) are needed to recycle the oxaloacetate because oxaloacetate has no mito-
chondrial transporter.
first discuss photosynthesis – a pathway that, like oxidative phosphorylation, extracts energy
and can be understood based on the chemiosmotic hypothesis.
SUMMARY
Oxidative phosphorylation is the major pathway for the extraction of energy from food.
There are two linked, vectorial flows: electrons from NADH to O2, and protons from the
mitochondrial matrix to the cytosol. These flows establish a proton gradient that leaves the
mitochondrial matrix with a lower concentration of protons and a negative electrical charge.
The return of protons to the matrix is coupled to ATP synthesis. Five protein complexes are
responsible for these activities, known as complexes I through V (as well as having more
descriptive names). The first four are electron carriers, and the fifth catalyzes ATP synthesis.
Each of the complexes is embedded in the mitochondrial inner membrane and is physically
isolated from the other four. Communication occurs exclusively through mobile cofactors.
NADH, UQ, and cytochrome c connect complexes I through IV. This flow can be depicted
as follows:

NADH ® éComplex I ù ® UQ ® éComplex III ù ® cyt. c ® éComplex IV ù ® O2
ë û ë û ë û
Additionally, electrons from succinate enter the pathway:
succinate ® éComplex II ù ® UQ
ë û
and the flow continues as in the first sequence, bypassing complex I.
Protons are produced by a variety of mechanisms: pumps in complexes I and IV, a loop
in complex III, and by annihilation (i.e., the removal of matrix protons into the hydrogens
of water) at complex IV. The electrical and chemical gradient of these protons is known as
the proton-motive force, which is the intermediate for energy generation. Complex V, also
known as ATP synthase, conducts protons back into the mitochondrial matrix space and
drives the synthesis of ATP from ADP and Pi through an unusual mechanism of rotating a
portion of the complex, an example of a molecular motor.
The linking of substrate oxidation with ATP formation is called coupling, and it is
expressed in the name oxidative phosphorylation. Some substances are uncouplers; that is,
they allow oxidation to proceed in the absence of ATP formation. Uncouplers are hydropho-
bic, weak acids that provide an alternative to proton re-entry to the mitochondrial matrix.
Beyond the protein complexes, there are membrane proteins of the inner mitochondria that
allow the selective exchange of several other substances, such as ADP, ATP, Pi, malate, aspar-
tate, and glutamate. Molecules that are not themselves transported, such as NADH, can use
indirect means – in some cases, making use of these transporters – to allow the entry of the
electrons from NADH (reducing equivalents) for use in mitochondrial oxidation.
The involvement of protons as the key energy intermediate is known as the chemiosmotic
hypothesis. It accounts not only for mitochondrial energy production but also for the capture
of light energy in photosynthesis.
REVIEW QUESTIONS
1. What is the proton-motive force?
2. What are the complexes of the inner mitochondrial membrane?
3. Which step of the Krebs cycle is also part of the electron transport chain?
4. How are the respiratory complexes related to the respiratory chain?
5. Suppose you were presented with the following sequence of electron flow:
NADH ® Fe-S ® FMN ® b ® Q.
Is this sequence possible? Explain.

6. What are the three ways that a proton gradient can be formed?
7. How does the loop mechanism work?
8. What is the role of flavin nucleotides in electron transport?
9. How is ATP synthesized?
10. How do uncouplers work?
11. Brown fat, rich in mitochondria, has recently been discovered in adult humans. This
tissue expresses a protein that causes uncoupling. If the tissue can be increased, it
would cause weight reduction. Why?
12. What is the mechanism for the regulation of ATP production by mitochondria?
13. Both the dissociated uncoupler (A-) and the ubiquinone radical ion UQ●− are delocal-
ized molecules. Yet, A- crosses the membrane and UQ●− does not. Explain.
14. How can Complex II and Glycerol P oxidase accept electrons from opposite sides of
the mitochondrial membrane, and yet both donate the electrons to the same mobile
cofactor, UQ?
CHAPTER 11 ADDENDUM: SUPERCOMPLEXES
An ongoing hypothesis among some structural biologists is that all of the complexes involved
in electron transfer are bound together as a supercomplex, such that there are no actual
mobile cofactors. The hypothesis suggests that UQ and cytochrome c are part of a supercom-
plex that can carry electrons directly from NADH to oxygen. Structural studies, including
the physical association of the purified components and electron microscopy studies, have
shown that the interaction can at least take place. As for why this association might have a
physiological basis, proponents offer two arguments: chemical efficiency and prevention of
oxygen radical formation.
The fact that enzyme complexes exist is undisputed; each of the four complexes are indi-
vidual examples. The question of whether a supercomplex is the functional expression of the
respiratory chain, however, is controversial.
262 Key Terms
While it might seem that chemical efficiency is a strong argument, it is a justification

rather than a requirement. There is no reference point for efficiency, so we are left with
attempting to compare rates of processes that are in complex versus individual reactions. For
one example, in Chapter 14, we will examine the pathway for fatty acid synthesis by mam-
mals, which is the ultimate in complex formation: seven reactions occur on the same protein.
The intermediate steps of each enzyme reaction are directly passed from one enzyme to the
next. Before we conclude that this is a marvel of efficiency for this pathway, consider this:
seven separate enzymes catalyze the same pathway in bacteria. It is not possible to argue that
bacteria are less efficient; the entire life span of bacteria can be less than one hour! It is dif-
ficult to imagine a living system more efficient than bacteria.
The argument that reactive oxygen species, a normal byproduct of portions of the respira-
tory chain such as Complex I, are minimized with complex formation is again an argument
without a baseline. Since these reactive species do actually form, how do we know that they
would be greater if the supercomplex exists or not?
We come down to the essentials by considering an objection some investigators have
raised to counter supercomplexes: with no mobile cofactors, how can electrons be passed
from both Complex I and Complex II? Proponents of the supercomplex hypothesis have
suggested that there are two pools of UQ, so that separate supercomplexes form and UQ
need not be a mobile cofactor. To understand the difficulty with this explanation, consider
the fact that UQ collects electrons not only from Complexes I and II but from several other
membrane-bound complexes, such as glycerol phosphate oxidase, choline oxidase, the elec-
tron-transferring flavoprotein of fatty acid oxidation, and dihydroorotate DH. To salvage the
supercomplex hypothesis, there would have to be at least six separate complexes of all of
these enzymes and correspondingly six separate UQ pools. The requirement for the extra
biosynthesis to accomplish this would be enormously inefficient.
There is another issue making the supercomplex notion difficult: the Q cycle itself. As
described, the mechanism for proton transport by Complex III requires movement of UQ
and UQH2 between the two faces of the mitochondrial inner membrane. That mobility is
precluded if we accept supercomplexes, and thus we are required to jettison that mechanism
and the studies that led to it. These are not trivial and would need alternative explanations for
the observations. While it is always possible that well-established concepts may be replaced,
merely suggesting that in vitro association of complexes rules them out is a weak argument.
Cytochrome c, by contrast to UQ, does not appear to connect multiple complexes.
However, it does play a separate role in cellular biology. During the process of apoptosis, it
is released from the mitochondria as the outer membrane pores are opened (a mitochon-
drial outer membrane permeability transformation), and cytochrome c enters the cytosol.
Once there, it becomes part of a complex that has protease activity leading to the ordered
destruction of the cell, or apoptosis. Suppose instead that cytochrome c was a tightly bound
component of a supercomplex. In this case, it could hardly be readily released to the cytosol.
In conclusion, the supercomplex is a hypothesis with some in vitro experimental support
but no clear physiological role for cells. The penchant for suggesting that complex formation
exists because of in vitro studies must be tempered by what we know of complex formation
in the first place: it serves to ensure that no side reactions are possible.
KEY TERMS
annihilation
apoptosis
binding change
chemiosmotic hypothesis
electrogenic
electron transport chain
glycerol phosphate shuttle
oxidative phosphorylation
proton-motive force
Q-cycle
reducing equivalents
respiration
shuttles
uncoupling
vectorial
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Photosynthesis 12
The carbon cycle is the global interchange of CO2 between animal production and plant
utilization. It is a topical issue: currently, much more CO2 is being produced than is being uti-
lized, so this cycle is out of balance. Global warming likely originates with the 19th-century
Industrial Revolution and the increased burning of fossil fuels. At that time, it was postulated
that an increase in atmospheric CO2 might cause increased global temperature. A steadily
rising global CO2 concentration was experimentally confirmed in the middle of the 20th
century.
Organisms that consume organic compounds and produce CO2 are auxotrophic. CO2
is converted back to organic compounds by photosynthetic organisms. Our focus in this
chapter will be on plants. A second cycle involves shuttling oxygen between its production
by plants and its consumption by animals.
Photosynthesis has two natural divisions: the light reactions, which include oxygen pro-
duction and photochemical events, and the carbon reactions (formerly dark reactions; see
the chapter addendum) by which CO2 is converted to organic compounds, such as sugars.
While we discuss them separately, both proceed simultaneously in plants.
12.1 LIGHT AND CARBON REACTIONS
The overall reaction of photosynthesis is:
(12.1) CO2 + H 2O + light ® CH 2O + O2
The unique feature of Equation (12.1) is the incorporation of light energy to drive carbohy-
drate production (CH2O) from CO2. A slightly more detailed view of this process exposes the
roles of the mobile cofactors ADP, Pi, ATP, NADP+, and NADPH:
(12.2) ADP + Pi + NADP+ + H 2O ® O2 + ATP + NADPH
(12.3) CO2 + ATP + NADPH ® CH 2O + ADP + Pi + NADP +
Equation (12.2) summarizes the overall light reactions, whereas Equation (12.3) summa-
rizes the overall carbon reactions. We have already encountered ATP, ADP, and Pi, mobile
cofactors that represent the transfer of phosphate bond energy. The pair of mobile cofactors,
NADP+/NADPH, was introduced in Chapter 7 as virtually identical in structure and func-
tion to NAD+/NADH. These pairs have the same standard redox potential, but very different
cellular redox potentials. In photosynthesis, NADPH represents electron-rich energy like
that of NADH in mitochondria.
Many of the individual light reactions are closely analogous to those of the mitochon-
dria. The same chemiosmotic hypothesis (i.e., production of a H+ gradient across a mem-
brane) explains the generation of energy in both cases. There are other remarkable analogies
between these processes, with the principle distinction being the input of light energy for
photosynthesis.
265
266 12.2 Chloroplasts
Carbon reactions utilize the cofactors produced by the light reactions – ATP and
NADPH – to incorporate CO2 into carbohydrates. Because both light and carbon reactions
take place within the chloroplast, the organelle unique to plant cells, we begin by examining
this structure.
12.2 CHLOROPLASTS
While mitochondria are found in both plants and animals, chloroplasts are a separate organ-
elle exclusive to photosynthetic organisms. We have noted previously that mitochondria
are evolutionary descendants of bacteria. Chloroplasts are descendants of cyanobacteria.
Chloroplasts retain their own (limited) genetic machinery and can carry out photosynthesis
successfully in isolation.
Like mitochondria, chloroplasts have an outer and inner membrane. However, there is a
further interior membrane, the thylakoid membrane. The physical arrangement is indicated
in Figure 12.1a. The thylakoid membrane appears in stacks known as grana, which allows
a large membrane surface area. Still, the interior volume of this membrane, the lumen, is a
single continuous space. A functional topology is presented in Figure 12.1b, showing a single
lumen space that is interior to the thylakoid membrane. The water space outside the thyla-
koid membrane, but inside the inner membrane, is the stroma. The thylakoid membrane
is analogous to the mitochondrial inner membrane because, in photosynthesis, all of the
membrane-bound proteins involved in light absorption and electron transport are embed-
ded in this membrane.
12.2.1 ORIENTATIONS: N AND P SIDES OF THE MEMBRANE
The chemiosmotic hypothesis applies broadly to mitochondria, chloroplasts, and bacteria.

The biological distinctions between these organelles can obscure their mechanistic unity;
that is, a proton gradient is formed across a membrane, leaving one side more deficient in
hydrogen ions. Thus, the mitochondrial inner membrane separates the matrix from the cyto-
sol, the thylakoid membrane separates the stroma from the lumen, and the bacterial cell
membrane separates the extracellular space from the cytosol.
FIGURE 12.1 The chloroplast. (a) Spaces and membranes of the chloroplast are indicated dia-
grammatically. The membrane holding the components of electron transport – the thylakoid
membrane – has extensive infoldings. Only a portion is indicated here (the open ends indicate
further membrane extensions, not an opening to the thylakoid lumen). (b) Functional diagram of
the chloroplast for the light reactions. Only the thylakoid membrane and its inner (lumen) and
outer (stromal) spaces are required.
Chapter 12 – Photosynthesis 267
FIGURE 12.2 Mapping positive and negative spaces across biological systems. While different
names are used in mitochondria, chloroplasts, and bacteria, the movement of a proton in each
case creates a positive and a negative side, universally labeled as the P and N sides.
This similarity becomes apparent from a designation for the space that is deficient in
protons – and thus negatively charged when a gradient is created – as the N side. The oppo-
site side, which is relatively positively charged, is called the P side. Figure 12.2 shows all three
energy-trapping systems and the corresponding labels for the N and P sides. For mitochon-
dria, the matrix is the N side; for chloroplasts, the stromal space is the N side; and for bacte-
ria, the cytosol is the N side, and its exterior is the P side.
12.2.2 LIGHT REACTIONS OF PHOTOSYNTHESIS AS A

REVERSED OXIDATIVE PHOSPHORYLATION
There is a strong analogy between the mitochondrial and the photosynthetic apparatus
for electron flow. Figure 12.3 shows both processes, with the omission of mitochondrial
Complex II (which leaves electron flow from NADH to O2 intact). Mitochondrial flow is
drawn from right to left. The three electron-transferring complexes of chloroplasts line up
with their counterparts in mitochondria (Figure 12.3). The photosynthetic central complex
b6f is analogous to mitochondrial Complex III. Both operate by the Q cycle mechanism,
which is a loop that moves electrons between membrane faces as protons move across the
membrane.
The mobile cofactors connecting the complexes are also similar. Plastocyanin (PC) is a
small redox-active protein that is mobile on the membrane surface, corresponding to mito-
chondrial cytochrome c. Plastoquinone (PQ) is a structural analog of ubiquinone (UQ)
H3 C CH3
H3C H
O CH3 9
Ubiquinone Plastoquinone
268 12.2 Chloroplasts
FIGURE 12.3 Comparison of photosynthetic and mitochondrial electron flows. The mito-
chondrial inner membrane (above) and thylakoid membrane (below) are aligned to highlight
their similarities. Note that electron flows are in opposite directions. The mobile cofactors, sub-
strates, and products are functionally and structurally similar. Central complexes are indicated as
ovals. PS I and PS II have the additional quality of light absorption to enable thermodynamically
allowed redox transfer.
and serves the same function. NADP+/NADPH is a cofactor pair nearly identical to NAD+/
NADH. The O2/H2O pair is the same, as it must be for the global cycling of oxygen between
plants and animals.
The other two complexes of the thylakoid membrane have similarities to mitochondria as
well. In each case, the protein complexes provide a pathway for electron transfer and react
with chemically similar mobile cofactors. The key distinction is that light is utilized in photo-
synthesis, so that understanding photosynthetic energy generation requires some knowledge
of photochemistry. Engineers interested in the artificial capture of solar energy also study
photosynthesis to improve the efficiency of their systems.
To appreciate the distinctions between mitochondria and chloroplasts, consider the anal-
ogy of Complex IV with photosystem II (PS II). Both complexes interact with the O2/H2O
pair. In the case of Complex IV, the active site contains Cu+ ions that strongly bind O2 (the
substrate) but weakly bind H2O (the product). Hence, mitochondria bind O2 well and release
H2O. On the other hand, the active site of PS II uses Mn2+ ions. Thus, chloroplasts bind H2O
well and release O2.
12.2.3 CARBON REACTIONS OF PHOTOSYNTHESIS: CO2 FIXATION
The carbon reactions bear considerable similarity to the pathways of glycolysis and the Krebs
cycle, in that they are metabolic sequences of enzymatic steps, with familiar patterns of elec-
tron rearrangement. However, one reaction is unique: the incorporation of CO2 into stable
organic molecules, the so-called “CO2 fixation”. In addition, the transketolase-catalyzed
reaction has a mechanism we have not encountered before, in which molecular fragments are
exchanged between substrates. Most of the reactions we will examine are part of the Calvin
cycle, which is the essential process of incorporating CO2 into a precursor to carbohydrates.
Portions of this pathway are very similar to the pentose phosphate shunt (Chapter 13). In
summary, the carbon reactions utilize the energy trapped in the intermediates ATP and
NADPH to synthesize sucrose and starch.
12.3 HARNESSING LIGHT ENERGY
Photochemical reactions are a departure from the chemical rearrangements we have consid-
ered so far. First, light itself is an energy form that depends upon its frequency, as expressed
by Equation (12.4):

(12.4) E = hv
where E is energy, h is the Planck constant, and ν is the frequency of the light. At a given
value of ν, the amount of energy, E, has a specific value; this package of energy is also called a
photon. The critical molecule engaged in trapping light energy is chlorophyll
which absorbs light in the blue and red regions of the visible spectrum (with wavelengths of
about 400 nm and 700 nm, respectively; wavelength is inversely proportional to frequency).
The green appearance of the chlorophyll pigment is due to the reflection of light at wave-
lengths between the absorption peaks (Figure 12.4).
FIGURE 12.4 Chlorophyll visible spectrum. The absorption spectrum of visible light for chlo-
rophyll-a shows that green light is not absorbed; its reflection accounts for the green color of
plants.
270 12.3 Harnessing Light Energy
Four distinct events occur in light-trapping:
1. The absorption of light by a large pool of chlorophyll molecules called antennae.

2. The transfer of the energy to adjacent chlorophyll molecules within the antenna.
3. The transfer of the energy to a special pair of chlorophyll molecules called the reac-
tion center.
4. The utilization of light energy to create a charge separation.
The last step can be alternatively viewed as boosting the redox level of an electron or, simply,
exciting it. Beyond this point, a series of redox transfers occur similar to those in oxidative
phosphorylation.
These processes take place in a protein complex called a photosystem (PS). Plants use two
photosystems, designated PS I and PS II. These were named in the order of their discovery;
in the actual sequence of electron transfer for photosynthesis, PS II occurs first. Nonetheless,
the essential features involved in light absorption and energy transfer are the same in each.
12.3.1 LIGHT ABSORPTION AND THE ANTENNAE
Light is trapped within a photosystem by the numerous chlorophyll molecules that form
the antenna system. There are a few hundred such chlorophyll molecules, any of which can
absorb a photon of light. The underlying theory for light absorption and its consequences
was developed in the early 20th century and forms the basis for quantum mechanics. The
energy transfer from light to electrons in chlorophyll occurs as discrete packages and at spe-
cific levels. Visible light has less energy (i.e., lower frequencies and longer wavelengths) than
the ultraviolet light that is absorbed by most organic compounds. However, the extensive
resonance of chlorophyll enables it to absorb visible light. When a molecule absorbs a spe-
cific wavelength of light, electrons are excited to a higher energy state; the entire molecule
is considered to be in an excited state. In an isolated molecule, the excited state returns to
the ground state (decays) with the release of energy in different ways such as fluorescence,
luminescence, or emission.
If the molecule is not isolated, upon excitation, the energy can be transferred to a very
close neighbor (i.e., about 15 Å away). In general, this is known as resonance energy trans-
fer. A specific form of this energy exchange that exists between chlorophylls is known as
exciton transfer.
There is a special pair of chlorophyll molecules that undergoes photooxidation; these are
the key parts of the reaction center. Reaction centers have associated proteins, quinones, and
pheophytins (structurally similar to chlorophyll molecules except that the chelate ring has
no metal inside it).
12.3.2 ELECTRON TRANSFER AT REACTION CENTERS
The unique feature of photosynthesis is the conversion of light energy to electron energy.
The reaction-progress curve shown in Figure 12.5 indicates the rapid change of redox poten-
tial energy following light absorption in PS II. This process is similar for both photosystems,
although there are distinct wavelengths for absorption, indicated as P680 for PS I and P700 for
PS II. In each case, the elevation of electron energy drives the ensuing redox chemistry, with
sequential reactions decreasing in redox potential, similar to those that occur in mitochondria.
Electron flow through the thylakoid membrane takes place in essentially the oppo-
site direction to that of the mitochondrial inner membrane. Overall, electrons from water
are removed, passed between protein complexes within the membrane, and terminate in
NADPH. During this process, protons move across the membrane, from the stroma to the
lumen (N to P). Three complexes of proteins embedded in the thylakoid membrane are
involved in photosynthetic chemiosmosis. The sequence of electron flow is:
H 2O ® PSII ® PQ ® b6 f ® PC ® PSI ( Fd ) ® NADP +

FIGURE 12.5 Energy diagram of light absorption. A reaction progress-midpoint potential dia-
gram illustrates the shift in E0 with the absorption of light.
FIGURE 12.6 Energetics of photosynthetic electron transport. Two light-driven steps form
part of the electron flow from H2O to NADPH. Cytochrome b6/f is an intermediate complex. The
activated states of the photosynthetic complexes are indicated with asterisks.
The components indicated in bold type are mobile entities that communicate between
the complexes. PQ (plastoquinone) is similar in structure and function to ubiquinone.
Plastocyanin (PC) is a redox protein containing a redox-active copper chelate analogous to
those in mitochondrial Complex IV. Ferredoxin (Fd) is another protein containing a redox-
active iron, mobile at the surface of the thylakoid membrane.
The input substrate is H2O, and the ultimate acceptor of electrons is NADP+. The com-
plexes of the electron transport chain (Figure 12.6) are PS II (photosystem II), the complex
b6f, and PS I (photosystem I). The mobile cofactor ferredoxin (Fd) has an alternative route
(cyclic photoelectron transport), which we will examine later.
The Z-scheme incorporates the light energy input (separately displayed for one photosys-
tem in Figure 12.5) with the chemical electron transfer reactions such as those in mitochon-
dria. The Z-scheme of Figure 12.6 displays the redox levels during the transfer of electrons
from H2O to NADP+. The diagram also shows the sequence of carriers and the striking dis-
tinction between light energy-boosting redox potential and non-light-driven electron flow,
272 12.4 Proton and Electron Flow for the Light Reactions
Box 12.1 Letter Origins: Behind the Z-Scheme
The term Z-scheme did not appear in the original graph of redox potentials of electron
carriers in photosynthesis in sequence. The first breakthrough in the understanding of
the energetics in chlorophyll was the finding that two photochemical centers in series
could drive electrons from a poor donor (H2O) to a weak acceptor (NADP+). Only later
was the redox potential–progress curve called a Z-scheme, displaying a combination of
thermodynamic and kinetic data in a way that is a pleasing counterpart to the represen-
tation for mitochondria. Rather than a steadily dropping potential, there are two abrupt
reversals in the Z-scheme, which represent energy input due to light absorption in the
two photosystems. Like the mapping of mitochondrial electron transport, the Z-scheme
is not a mechanism but rather an energy outline of the data for the sequence of electron
flow. Because some photosynthetic organisms have just one photosystem, the Z part
is itself dispensable. Curiously, the name enjoys universal acceptance even though the
overall shape is more of an “N” than a “Z.” The designation is a historical accident; early
representations had reversed axes, so the original plot was in a Z shape. Nonetheless,
the scheme does preserve the connection between redox potential and photosynthetic
electron flow, with the introduction of just one new element: light energy.
decreasing redox potential. The activated states of the P680 and P700 chlorophylls are indi-
cated with asterisk superscripts; this is the result of electrons within these molecules being
boosted by light absorption. The subsequent transfer of electrons, as noted, leads to a drop
in free energy. As an aside, the origin of the name “Z-scheme” stems from the fact that the
original diagram had its axes reversed (Box 12.1).
12.4 PROTON AND ELECTRON FLOW FOR THE LIGHT REACTIONS
The movement of electrons from H2O to NADPH, coupled to proton flows and ATP syn-
thesis in the chloroplast, is illustrated in Figure 12.7. The electron sequence is now placed in
the context of the thylakoid membrane. Electron flows between the complexes are mediated
by the mobile cofactors PQ (plastoquinone), which is mobile within the lipid phase of the
thylakoid membrane, and PC (plastocyanin), which is mobile on the surface of the thylakoid
membrane. The cyt b6/f complex is the only site where protons cross the membrane, using
a loop mechanism (Figure 12.8), closely analogous to that of mitochondrial Q-c reductase
(Complex III). Once again, electrons travel across the membrane through a pair of b cyto-
chromes, and a lipid-soluble quinone (here, PQ) moves across the membrane alternately in
reduced and oxidized forms.
A further contribution to the proton gradient is the conversion of H 2O to O2 and H+ at the
lumen face of PS II (Figure 12.7). The event is the reverse of annihilation in mitochondria.
In photosynthesis, protons are created in the lumen (the interior space), making the stromal
space relatively more alkaline.
Utilization of the proton gradient also mirrors the mitochondria, in that the CF1CFoATPase
has a virtually identical mechanism to the F1FoATPase, with the same molecular motor
mechanism.
The overall proton-motive force (Δp), which represents the sum of the electrical and chem-
ical gradients of the proton, is also very similar between mitochondria and chloroplasts, with
each having a value of about 200 m V. However, the chloroplast has less need for an electri-
cal gradient. There are no electrogenic transfers needed across the thylakoid membrane. The
principal electrogenic transporter in the mitochondria, ATP/ADP exchange, is not present in
the thylakoid because ATP is generated outside the lumen (on the N side). The initial genera-
tion of Δp is almost entirely electrical but subsequently is dissipated by counter ion move-
ments (largely Mg2+). While about half of the resulting Δp remains as an electrical gradient,
the chloroplast is capable of maintaining a large chemical gradient of up to three pH units.
FIGURE 12.7 Electron and proton flows in photosynthesis. Electrons flow through the three
complexes, culminating in NADPH formation. PS II and the cytochrome b6/f complexes contrib-
ute to the creation of the proton-motive force, which is utilized in ATP formation.
FIGURE 12.8 Cytochrome b6/f loop mechanism. The movement of electrons through the
complex to the ultimate acceptor, plastocyanin (PC), as well as cycling through the complex via
two b cytochromes, is closely analogous to the Q–cytochrome c complex of mitochondria. The
fully reduced and fully oxidized forms of plastoquinone move across the membrane, ferrying
protons to create a proton-motive force, the same role played by ubiquinone in mitochondria.
274 12.5 Cyclic Electron Transfer and Other Variations
FIGURE 12.9 Cyclic photosynthetic electron flow. Two of the three complexes of the thylakoid
membrane are involved in the cyclic flow. Electrons flow from the b6/f complex to PC and then
to PS I. Electrons also are delivered to the mobile cofactor ferredoxin (Fd), but, rather than reduc-
ing NADP+, they travel instead back to the b6/f complex, completing a cycle. Note that the loop
mechanism is still functional, so that a proton-motive force is still created, and ATP formation
proceeds in the absence of NADPH formation.
12.5 CYCLIC ELECTRON TRANSFER AND OTHER VARIATIONS
Under some conditions, an abbreviated pathway of electron transfer takes place, which
makes use of only PS I and the b6f complex (Figure 12.9). This route makes use of the mobile
nature of ferredoxin. Instead of providing electrons to ferredoxin reductase (and then to
NADPH), ferredoxin can move on the stromal surface of the membrane and donate electrons
to the cytochrome b6f complex. Thus, PS I replaces PS II as an electron donor to reduce the
central complex (b6f). Electron transfer is to PC, then to PS I, forming a cycle. The proton
gradient is still produced – the loop remains – and ATP can be synthesized. However, PS II
is not utilized, so O2 is not produced. Because ferredoxin reductase is bypassed, NADPH is
not produced either. We may view the cyclic pathway as a means of producing ATP without
NADPH formation, providing metabolic flexibility.
The linear path from H2O to NADPH – sometimes called the noncyclic pathway – is
present in many organisms from algae to higher plant forms, but variations exist. For exam-
ple, purple photosynthetic bacteria have just one photosystem. The presence of grana stacks
is not an essential part of chloroplast structure either. When grana are not present, the chlo-
roplast contains only PS I, not PS II. The PS I complex does not occur within membrane
stacks, probably because this complex must connect to the membrane surface to accommo-
date the mobile cofactors PQ and ferredoxin.
At the molecular level, there is variation among plants in both chlorophyll subtypes and
accessory pigments, such as the carotenoids. While chlorophylls number in the hundreds
for each photosystem, the carotenoids are present at a level an order of magnitude lower. The
function of carotenoids is twofold. First, as they absorb light at distinct wavelengths from
chlorophyll (which is evident from their distinctive reddish-orange color), they can trap light
from other parts of the electromagnetic spectrum. The energized carotenoids excite to higher
levels and can pass this energy to chlorophyll molecules within the antenna. Secondly, carot-
enoids serve as antioxidants, preventing damage to chlorophylls from reactive oxygen species.
12.6 THE CALVIN CYCLE
Melvin Calvin proposed a cyclic pathway for the incorporation of CO2 into organic com-
pounds. Like the Krebs cycle, the Calvin cycle has a definite substrate and a definite product.
For the Calvin cycle, the substrate is CO2, and the product is glyceraldehyde-3-P (GAP).
Carbon flow in the cycle – ignoring stoichiometry at first – is outlined in Figure 12.10. A
C5 sugar reacts with CO2 to form two C3 molecules. After phosphorylation (using ATP) and
reduction (using NADPH), the result is glyceraldehyde 3-P (GAP). Of the GAP produced,
one molecule exits the cycle – this is the product – and the rest continue until the C5 is
regenerated.
Most of the reaction mechanisms are similar to those we have encountered before. One
new type (transketolase) also appears in the pentose shunt, which we discuss in Chapter 13.
The reaction step unique to the Calvin cycle is the incorporation of CO2, which is also the
first reaction of the pathway.
12.6.1 RIBULOSE BISPHOSPHATE CARBOXYLASE
Commonly abbreviated RuBisCo, ribulose bisphosphate carboxylase is the most abundant

enzyme in the world. In the chloroplast, it can account for as much as 15% of the total protein
content. It is unique in being able to incorporate net CO2 into organic products. No compa-
rable reaction occurs in the animal world.
In the RuBisCo reaction (Figure 12.11), CO2 reacts with the five-carbon molecule ribulose
bisphosphate to produce two molecules of 3-P-glycerate (PGA). The mechanism (Figure 12.12)
involves an enediol intermediate, similar to the one in the isomerase reactions that we have
examined elsewhere (e.g., glucose phosphate isomerase and triosephosphate isomerase in
Chapter 9). Just as we observed with the isomerase enzymes, RuBisCo produces this enediol
intermediate through proton abstraction and electron rearrangement.
The enediol intermediate (Figure 12.12a) forms a carbanion; the electron pair of this
carbanion attacks the carbon of CO2 to form the 2-carboxy-3-keto intermediate. In the
second portion of the mechanism (Figure 12.12b), the reaction proceeds in a similar way
to the lysis direction of the aldolase reaction. The addition of water to the carbonyl leaves
FIGURE 12.10 Carbon flow in the Calvin cycle. Molecules are represented as strings of filled
circles (carbon atoms). The sole input to the cycle is CO2; the output molecule is GAP (glycer-
aldehyde P). CO2 reacts with a 5-carbon substrate of the cycle to produce two 3-carbon cycle
intermediates (PGA). Subsequent reactions to GAP consume ATP and NADPH. One GAP leaves
the cycle (as product); the remainder are converted back to the 5-carbon intermediate that once
again reacts with a CO2 molecule to continue the cycle.
276 12.6 The Calvin Cycle
FIGURE 12.11 The RuBisCo reaction. Incorporation of CO2 is catalyzed by ribulose-bisP car-
boxylase (RuBisCo). The carbon of the CO2 is labeled to show its point of entry into one of the
two P-glycerate molecules.
FIGURE 12.12 Mechanism of RuBisCo. The mechanism of RuBisCo is divided into two parts.
(a) The removal of a proton and formation of dienol, which attacks the CO2 to form carboxy-
ribulose-bis-P. (b) The hydrolysis of this intermediate, followed by electron rearrangement, which
leads to its cleavage into two molecules of PGA.
a negatively charged oxygen, subsequent rearrangement of electrons splits the C–C bond,
forming two triose phosphates. After a protonation step, both products are the same mol-
ecule: PGA.
12.6.2 REACTION STEPS FOLLOWING CARBON FIXATION TO

GLYCERALDEHYDE-P: ENERGY CONSUMING PORTION
The steps following the incorporation of the CO2 are similar to the reactions of glycolysis.
The transformation of PGA to GAP uses ATP and NADPH, the energy molecules formed
during the light reactions. The reactions are shown in Figure 12.13.
The first reaction after CO2 incorporation is catalyzed by P-glycerate kinase, which
proceeds in the reverse direction to glycolysis. The next reaction is catalyzed by an
NADP+-linked glyceraldehyde-P-DH, virtually identical to the glycolytic enzyme, which
uses NAD+. At this point, the product of the Calvin cycle – GAP (glyceraldehyde-P) – is
produced. For every three CO2 molecules incorporated by RuBisCo, one GAP molecule
is formed. However, in the stoichiometric balance of the cycle, six molecules of GAP are
produced during this reaction sequence, and only one of those represents the product.
The remaining GAP molecules continue the cycle to form RuBP through 10 reaction
steps.
12.6.3 FROM GAP TO THE RUBP: OVERVIEW
While GAP is a product of the cycle, it is also an intermediate of the cycle. Both roles are
illustrated in the stoichiometry outline of Figure 12.14. The portion labeled (1) consists of
RuBisCo and the reactions just discussed that convert RBP into GAP. As indicated, for every
3 molecules of CO2, one molecule of GAP leaves the cycle. Because process (1) actually pro-
duces 6 molecules of GAP, the remaining five continue through the Calvin cycle and are
ultimately converted into 3 molecules of RBP (3C5), that is, process (2).
Restating our stoichiometry, process (2) converts 5 molecules of C3 into 3 molecules of C5.
A shuffling of carbon fragments achieves the carbon balance through aldolase type reactions
and a new enzyme type, the transketolase. A more detailed outline of the stoichiometry of
the process is displayed in Figure 12.15. The aldolase type reactions are indicated as dotted
arrows. The transketolase reactions are shown as broad arrows. Notice that the transketolase
reactions require moving fragments of one molecule onto the other.
Three asterisks appear in Figure 12.15, indicating extra enzymatic reactions (omitted from
the diagram) that do not affect the stoichiometry. The molecules undergoing reaction are dis-
played in Figure 12.16. The reactions themselves are numbered in Figure 12.16 to facilitate
the discussion of the enzymatic steps in more detail.
FIGURE 12.13 Formation of glyceraldehyde P (GAP). GAP is formed from ribulose-bis-P in

the first three steps of the Calvin cycle. RuBisCo catalyzes the first. The next two enzymes are
P-glycerate kinase and GAPDH, which are very similar to those of glycolysis. Note that ATP and
NADPH, the energy molecules produced in the light reactions, are utilized here.
FIGURE 12.14 Stoichiometry of the Calvin cycle: overview. The balance of the Calvin cycle is
illustrated with most reactions omitted. In section (1), three CO2 molecules enter, and the cycle
produces one net molecule of the C3, GAP. Five other C3 molecules of GAP are converted by the
reactions of section (2) to three molecules of the C5 RuBP.
FIGURE 12.15 Stoichiometry of the Calvin cycle: recycling from C3. Carbon balance through
the Calvin cycle is further detailed here. The six C3 molecules are initially PGA, but they are con-
verted to GAP through the reactions shown in Figure 12.13. One GAP molecule is the product of
the cycle; the five others regenerate the three C5 molecules of RuBP. The thick, curved reaction
arrows represent transketolase reactions. The dotted reaction arrows represent aldolase reac-
tions. Reactions indicated by an asterisk undergo an additional reaction not shown here that
does not alter the carbon number.
12.6.4 FROM GAP TO THE RUBISCO STEP: REACTIONS
We now consider the reactions constituting the regeneration RuBP, the RuBisCo substrate,
from GAP. The reactions themselves are very similar to ones we have encountered before
except for a single enzyme type: the transketolase.
The first four reactions are displayed in Figure 12.17. These are:
(1) Triosephosphate isomerase reaction catalyzes the conversion of GAP to dihydroxy-

acetone-P (DHAP).
(2) The reaction of GAP with DHAP to form fructose-1,6-bisP (FBP), catalyzed by aldol-
ase, is the same as the glycolytic reaction.
FIGURE 12.16 Regeneration steps of the Calvin cycle. The reaction steps for converting the
five GAP molecules to RuBP involve transketolases (broad, curved arrows) as well as aldolases
(dotted arrows). Each reaction is numbered and detailed in Figures 12.17 and 12.19.
FIGURE 12.17 The Calvin cycle reactions from GAP to the first transketolase step: (1) Triose
isomerase (2) Aldolase (3) Fructose bisphosphatase (4) Transketolase.
(3) The enzyme fructose bisphosphatase catalyzes the dephosphorylation of FBP. The
reaction is a hydrolysis of the phosphoryl group. This step also appears as a late step
in glucose formation (Chapter 13).
(4) The conversion of F6P and GAP to E4P (erythrose-4P) and Xu5P (xylulose-5P) is
catalyzed by transketolase. As suggested by the name, this reaction results in an
exchange of ketone-containing molecules. The top portion of the F6P molecule is
outlined with a dotted boundary, indicated the fragment removed in the course of
the reaction and transferred to the second substrate, GAP. The products are E4P
(erythrose-4P, the C4 fragment that remains from F6P), and Xu5P (xylulose-5P),
formed by joining the C2 fragment from F6P with GAP.
The enzyme mechanism of transketolase is presented in Figure 12.18. As the mecha-

nism is symmetrical, only the first half is displayed. Like two other enzymes we have
encountered – pyruvate decarboxylase (Chapter 9) and the El of the pyruvate dehydrogenase
FIGURE 12.18 Mechanism of transketolase. The enzyme-bound thiamine facilitates the

removal of a two-carbon fragment of the substrate. Electron flow is similar to pyruvate decarbox-
ylase (Chapter 9) and E1 of pyruvate DH complex (Chapter 10), using the thiamine as carbanion
and electron sink. In the second half (not pictured), the fragment is donated to the carbonyl of
the acceptor molecule (here, GAP).
complex (Chapter 10) – transketolase is bound to the cofactor thiamine pyrophosphate. The
thiamine group is shown as the carbanion, which attacks the carbonyl of F6P and forms an
adduct. In the next step, a base from the enzyme removes the hydroxyl proton at C3 of the
substrate. An electron rearrangement follows, leading to a split in the C–C bond between
C2 and C3. At this point, E4P is released, leaving the two-carbon fragment attached to the
thiamine cofactor of the enzyme. The resonance form shown contributes to the stability of
this intermediate. The remainder of the mechanism involves binding to the second substrate,
GAP, attacking the carbonyl with the anion form, and, ultimately, releasing the free enzyme
and X5P, the joined five-carbon product. With this reaction, we can appreciate the versatility
of the thiamine cofactor, which participates in a decarboxylation (pyruvate decarboxylase),
the transfer to a separate nucleophile (pyruvate dehydrogenase complex El), and the carbon
shuffling reaction shown here.
The six remaining steps leading to RuBP formation are illustrated in Figure 12.19. These
reactions are also similar to ones we have already studied, and the sequence displayed also
contains a transketolase step. The reactions are:
(5) An aldolase catalyzed reaction, joining E4P with DHAP, leading to the formation of
the seven-carbon sugar SBP (sedoheptulose-1,7-bisP).
(6) The hydrolytic removal of one of the phosphoryl groups of SBP to form S7P, cata-
lyzed by a phosphatase. The reaction is analogous to the phosphatase reaction of
step 3.
(7) A second transketolase reaction, catalyzing the reaction of S7P with GAP, produc-
ing R5P and X5P.
(8) An epimerase enzyme catalyzes the conversion of Xu5P to the Ru5P. Another epim-
erase is featured in the catabolism of galactose (Chapter 13).
(9) An isomerase enzyme, with the same mechanism as the glycolytic isomerase
enzymes, catalyzes the conversion of R5P to Ru5P.
(10) A kinase catalyzes the conversion of Ru5P to the RuBisCo substrate, RuBP.
Another view of the regenerative reactions between GAP and Ru5P of the Calvin cycle is
offered in Figure 12.20. The figure uses a three-dimensional representation of a cube to
FIGURE 12.19 The Calvin cycle reactions from E4P and DHAP to RuBP. Numbering continues
from Figure 12.17, and corresponds to the overview of Figure 12.16. (5) Aldolase (6) Sedoheptulose
bisphosphatase (7) Transketolase (8) Xylulose-P epimerase (9) Ribose-P isomerase (10) Ribulose-P
kinase.
FIGURE 12.20 Calvin cycle regeneration reactions in 3D. Reactions of the regeneration phase
of the Calvin cycle are drawn on the surface of a cube to avoid the repetition of reaction sub-
strates. GAP entry in the middle corner of the cube is involved in several reactions, as is DHAP in
one of the cube faces. Reaction numbers correspond to Figure 12.16.
indicate the transforms that repeatedly use GAP and employ two transketolase reactions
(4 and 7).
Having considered the individual reactions, we can now step back and reconsider the over-
view of the Calvin cycle (Figure 12.14). The reactions represented by (1) start with RuBisCo,
the incorporation of carbon from CO2 into PGA, ultimately leading tothe formation of GAP.
One of the GAP molecules leaves the cycle, enters plant intermediary metabolism, and forms
all of its carbon structures. The pathway for the remaining GAP molecules is represented by
(2) in Figure 12.14 complete the Calvin cycle through the 10 reactions we have just detailed.
The ability of 5 three-carbon molecules to form 3 five-carbon molecules involves joining
reactions (aldolases) and fragment transfers catalyzed by transketolases. As with the Krebs
cycle, the intermediates can also be used in other reactions of plant metabolism.
It is important to emphasize the pathway substrate and product of the Calvin cycle.
The pathway substrate is CO2, and the pathway product is GAP. Only one of the GAP
282 12.7 Variations in CO2 Handling: C3, C4, and CAM Plants
molecules formed arises directly from the input CO2 (Figure 12.11). The overall pathway
is another example of repetition–variation, meaning that it is only in the steady-state,
with many turns of the cycle, that the overall conversion takes place in a stoichiometric
manner. Thus PGA carries the “fixed carbon” derived from atmospheric CO2 into sucrose
and starch.
12.7 VARIATIONS IN CO2 HANDLING: C3, C4, AND CAM PLANTS
Most plants fix CO2 by the process described in Section 12.6. Because the product of the fixa-
tion reaction has three carbons (PGA), these plants are also known as C3 plants. However,
there is a built-in inefficiency because RuBisCo also catalyzes its reaction with O2 in place of
CO2 (Figure 12.21). O2 can add to the enediol intermediate of RuBP (Figure 12.18), produc-
ing PGA and P-glycolate. While the Km for O2 is much greater than the Km for CO2, the far
greater concentration of atmospheric O2 (21% in air vs. 0.04% for CO2) means that a signifi-
cant amount of O2 can substitute for CO2. In a few cases in which the climate magnifies the
loss of carbon flow through the Calvin cycle, some adaptations minimize this loss by the
presence of additional reactions.
In the case of plants originally found in tropical climates, such as sugar cane and corn,
incorporation of CO2 involves a four-carbon rather than a three-carbon intermediate.
Accordingly, these organisms are called C4 plants. An example of this pathway is illus-
trated in Figure 12.22. Photosynthesis is divided between two cell types in these plants;
the bundle sheath cells and the mesophyll cells. The bundle sheath cells are arranged in
an inner ring around the vascular system of the plants. An outer ring contains mesophyll
cells. The Calvin cycle enzymes are located exclusively in the bundle sheath cells.
After CO2 enters the mesophyll cell from the exterior, it is converted to HCO3−, a step
catalyzed by carbonic anhydrase. Next, PEP carboxykinase catalyzes the reaction of HCO3−
with PEP to form oxaloacetate (OAA). As we will appreciate when we complete this path-
way, this carboxylation reaction is fundamentally different from RuBisCo in that CO2 is
ultimately released in the pathway. OAA is converted next to malate in a reaction catalyzed
by NADP+-linked malate dehydrogenase. Malate is then transported to the bundle sheath
cell by a transport protein that spans both cell membranes (plasmodesmata). Once in the
bundle sheath cell, malate undergoes decarboxylation to pyruvate and CO2, catalyzed by
malic enzyme. This reaction shares a mechanism with isocitrate dehydrogenase of the Krebs
cycle (Chapter 10). The pyruvate is transported back to the mesophyll cell (through a distinct
plasmodesmata), and converted to PEP in a reaction catalyzed by a dikinase. Regeneration of
PEP completes the cycle, which functions in producing augmented levels of CO2 for RuBisCo
so that the reaction with O2 is minimized. Reactions similar to this shuttle are in the path-
ways of gluconeogenesis (Chapter 13) and fatty acid synthesis (Chapter 14).
FIGURE 12.21 RuBisCo reaction with O2 in place of CO.

FIGURE 12.22 The C4 pathway in plants. The pathway involves two separate cells: the meso-
phyll and bundle sheath cells. The two are connected by transporters that span both cell mem-
branes. Upon entry to the mesophyll cell, CO2 is hydrated to HCO3− (1), a reaction catalyzed by
carbonic anhydrase. Next, PEP reacts with HCO3− to form oxaloacetate (OAA), catalyzed by PEP
carboxykinase (2). Following reduction of OAA to malate, catalyzed by an NADPH-linked malate
DH (3), malate is transported (4) into the bundle-sheath cell. Malate is converted to pyruvate and
CO2 through a reaction catalyzed by malic enzyme (5). The CO2 is the Calvin cycle substrate, and
the pyruvate is transported (6) back to the mesophyll cell and converted to PEP through the
pyruvate dikinase catalyzed reaction (7).
A further variation exists in plants that thrive in extremely hot, dry climates. As
these plants are succulents of the Crassulaceae family, they are called CAM plants, for
Crassulacean acid metabolism. Similar to C4 plants, an intermediate carboxylation occurs,
using the same enzymes – PEP carboxylase and malate dehydrogenase – to convert PEP to
oxaloacetate. In CAM plants, however, the separation of the parts of the cycle is accom-
plished in time rather than in space. At night, the cell admits air-containing CO2 and stores
malate, allowing entry through openings of pores to the cell called stomata. In the daytime,
the stomata close, and malate is decarboxylated to generate CO2 for the Calvin cycle.
Beyond these specializations, there are numerous minor pathways in specific plant
species as well as in microorganisms, such as bacteria and yeast. The products of these
pathways, known as secondary metabolites, have no obvious functional except per-
haps for their protection against consumption by predators. There is medical interest
in these pathways, because their products often selectively kill pests with few adverse
effects on humans. As a result, many drugs are derived from secondary metabolic
pathways.
284 12.8 Pathway Endpoints: Sucrose and Starch
12.8 PATHWAY ENDPOINTS: SUCROSE AND STARCH
GAP that arises from CO2 fixation can be converted to either sucrose or starch. Sucrose
is formed by transporting GAP from the chloroplast to the cytosol, followed by a series of
enzymatic steps leading to condensation of an activated glucose moiety and fructose-6-P.
Starch is formed by the sequential condensation of a distinct activated glucose moiety with
pre-formed glucose polymers. These “activated glucose” moieties are nucleotide sugars.
Both sucrose and starch can be used as energy precursors in plants for the generation of
ATP by pathways discussed elsewhere in this text. The two have roles analogous to glucose
and glycogen in animals, in the sense that sucrose is a mobile energy sugar that can be trans-
ported between cells through the plant vascular system, while starch is a storage form of
energy that is local to cells.
Sucrose is a nonreducing sugar, which means that there is no free aldehyde moiety in
equilibrium with the molecule. It is, therefore, more stable than glucose and better suited as
a sugar transport form in plants. However, it does require more enzymatic reactions for its
formation and breakdown and, thus, is not as readily available as glucose.
In the ensuing chapters of this text, we consider the remaining critical pathways of mam-
mals: other carbohydrate routes, fat metabolism, and nitrogen metabolism. Nonetheless, the
pathways taken by plants can be considered a superset of those for mammals. An analogous
photochemical reaction in humans is in the visual cycle, where the key molecule employed is
carotene, one of the accessory pigments of photosynthetic organisms (Box 12.2).
Box 12.2 Plants and the Visual Cycle
The first part of the visual cycle in humans and other animals is similar to photosynthesis.
For both processes, an embedded pigment bound to a membrane protein absorbs light,
and the energy of that absorbed light drives a reaction. In the visual cycle, the reaction
is the isomerization of a cis double bond in retinal to the trans configuration. In turn, this
changes the conformation of the bound protein opsin, which transmits the light through
a series of steps involving membrane polarization and neurotransmitter release from the
retina to the optic nerve. There are other similarities. The origin of human retinal is dietary
carotene (see the structures in Figure B12.2a), an accessory plant photosynthetic pig-
ment. The aldehyde of retinal forms a Schiff base with a lysine residue opsin; this adduct
is known as rhodopsin. Not only does the retinal portion alter the conformation of opsin
after its conversion to the trans form, but opsin influences the light absorption properties
of the bound retinal. In the cone cells of the retina, three different opsin proteins exist,
each of which senses specific bands of the light spectrum, accounting for sensitivity to
blue, green, and red light. Finally, the membrane arrangement of the light-absorbing pig-
ment in the eye resembles the grana stacks. Rhodopsin exists in dense stacks in internal
membranes of the outer segment of the rod cells (Figure B12.2b).
FIGURE B12.2A β-Carotene and retinal

FIGURE B12.2B A rod cell. On the left, a drawing of a rod cell showing the inner and outer
segments. On the right is an electron micrograph showing a detail of the rhodopsin stack in
the outer segment. (Reprinted from Histology of the Human Eyes, M. J. Hogan, J. A. Alvarado,
and J. E. Weddell, p. 425. Copyright 1971, with permission from Elsevier.)
SUMMARY
Photosynthesis can be divided into two separate processes: the light reactions, in which
energy is formed, and the carbon reactions, in which energy is used to convert CO2 to sugars.
The intermediate energy forms are ATP and NADPH, and they are employed in the carbon
reactions. Photosynthesis takes place in the plant chloroplast, a specialized organelle not
found in animal cells. Like the mitochondria, the chloroplast arose evolutionarily from bac-
teria. The pathway for energy formation involves light absorption by chlorophyll molecules
that is used to excite electrons and move them through a redox chain in close analogy to
mitochondria (run in reverse), from H2O to NADPH. The generation of a proton gradient is
consistent with the chemiosmotic hypothesis, which also accounts for mitochondrial and
bacterial energy formation. The use of the energy molecules ATP and NADPH in the Calvin
cycle drives the conversion of CO2 into sugar (C6H12O6); the Calvin cycle converts three
CO2 molecules into one of PGA (P-glycerate). In the carbon fixing reaction, ribulose-bis-P-
carboxylase (RuBisCo) catalyzes the incorporation of CO2 into ribulose-5-P, producing two
PGA molecules. PGA is recycled back to ribulose-5-P using several enzymatic reactions,
most of which are very similar to ones encountered previously. An exception is transketolase,
which catalyzes two separate steps of the pathway, in which carbon fragments are reshuffled
to convert one ketone molecule into another. The GAP exiting the Calvin cycle is converted
to sucrose for transfer to other parts of the plant, or to starch for energy storage. Variations
exist in some plants to concentrate CO2 (C4 plants) or to prevent water loss (CAM plants).
Other metabolic routes exist within many plant species to make a wide array of distinctive
molecules, some of which have significant medical uses.
REVIEW QUESTIONS
1. The two absorption maxima of light were given as 400 and 700 nm, at the red and blue
ends of the spectrum. Why are plants green?
286 Chapter 12 Addendum: Dark vs Carbon Reactions
2. While all plants would benefit from the use of the C4 route, most do not use it. What
steps of the C4 pathway require extra energy formation by the plant? How would that
affect global warming?
3. Other accessory pigments in plants account for the autumnal colors, mainly orange
and reds. One of these was identified in this chapter as carotene. Why should those
molecules persist in leaves while chlorophyll, which is typically far more abundant,
does not?
4. What is the principal advantage of energy transfer by exciton transfer in the antenna
complex versus direct absorption at the reaction center?
5. In terms of overall pathway consequence, distinguish CO2 incorporation by RuBisCo
from CO2 incorporation by PEP carboxylase in C4 plants.
6. Consulting Figure 12.11, CO2 is incorporated into one of the product molecules
of RuBisCo. Why does this imply that the Calvin cycle is another example of
repetition–variation?
CHAPTER 12 ADDENDUM: DARK VS CARBON REACTIONS

The division of chloroplast function between light and dark reactions has a pleasing sym-
metry. Without knowing the details of the reactions, we might imagine that the plant is busy
capturing light in the daytime and synthesizing sugar at night. This image is an unfortunate
consequence of the terminology.
Originally, carbon fixation and carbohydrate synthesis reactions were observed to occur
in the absence of light, provided ATP and NADPH were added externally in experimental
systems. The experiment suggested the division between light and dark. The intermediates
ATP and NADPH were of necessity produced by the chemiosmotic mechanism. Once those
energy intermediates were available, Calvin cycle and further synthetic reactions could pro-
ceed without the need for light energy. The only caveat offered was that these reactions did
not need to be in the dark; they simply did not require light.
The light–dark division is fundamentally erroneous. The intermediates ATP and NADPH
are not energy stores in plants any more than they are in animals. They do not accumulate;
rather they are transient molecules for the transfer of energy. Experimentally, we can supply
a preparation with these cofactors, but their concentration in cells is not regenerated and
would support biosynthesis for just a few seconds.
It is also difficult to reconcile the fact that several of the so-called “dark reactions” require
the conditions established by the light reactions. For example, pH optima of several biosyn-
thetic enzymes lie in the alkaline region. The pH shift resulting from chemiosmosis in the
chloroplast respiratory chain establishes this pH.
Due to the misleading nature of naming the biosynthetic pathway the dark reactions,
plant scientists have adopted the term carbon reactions. In the scientific literature, at least,
this has been steadily displacing the older designation over the last few decades.
Physiologically, at night plants do have an alternative for energy formation: the mitochon-
dria. Of course, this also means that, in the dark, plants consume O2 and produce CO2.
While this may appear on the surface to be a problem for those who have indoor plants, the
amount of O2 consumption and CO2 production by house plants at night is trivial.
KEY TERMS
antennae
auxotrophic
bundle sheath
C3 plants
C4 plants
Calvin cycle
CAM plants
carbon reactions
carbon fixation
chloroplast
exciton transfer
ferredoxin
fructose bisphosphatase
grana
light reactions
luminal
mesophyll
pheophytins
photosynthetic
photon
photosystem I (PSI)
photosystem II (PSII)
plasmodesmata
reaction center
rhodopsin
stroma
thylakoid membrane
transketolase
BIBLIOGRAPHY
C. Bathellier, G. Tcherkez, G.H. Lorimer, G.D. Farquhar. Rubisco Is Not Really So Bad. Plant Cell
Environ 41 (2018) 705–716.
B.B. Buchanan. The Carbon (Formerly Dark) Reactions of Photosynthesis. Photosynth. Res. 128 (2016)
215–217.
P. Fromme, P. Jordan, N. Krauss. Structure of Photosystem I. Biochim. Biophys. Acta 1507 (2001) 5–31.
D.O. Hall, K.K. Rao, B. Institute of Photosynthesis. Cambridge University Press, Cambridge. 2010.
A.U. Igamberdiev, P. Gardeström. Regulation of NAD- and NADP-Dependent Isocitrate
Dehydrogenases by Reduction Levels of Pyridine Nucleotides in Mitochondria and Cytosol of
Pea Leaves. Biochim. Biophys. Acta: Bioenerg. 1606 (2003) 117–125.
O. Morton. Eating the Sun: How Plants Power the Planet. Fourth Estate, London. 2009.
C.W. Mullineaux. Function and Evolution of Grana. Trends Plant Sci. 10 (2005) 521–525.
D.A. Walker. The Z-Scheme--Down Hill All the Way. Trends Plant Sci. 7 (2002) 183–185.
Carbohydrate Pathways
Related to Glycolysis 13
The pathways discussed in the present chapter will reveal the richness of carbohydrate
metabolism, showing new connections to the glycolytic pathway. Together with the prin-
cipal routes of lipid and nitrogen metabolism, we have a set of reactions collectively called
intermediary metabolism.
Here we consider several pathways that are connected to glycolysis. Figure 13.1 shows sev-
eral glycolytic intermediates as points of intersection for five routes of carbohydrate metabo-
lism. The first two, glycogen synthesis and glycogenolysis, will serve as a model for introducing
regulation in metabolism. Galactose catabolism intersects glycogen metabolism in the for-
mation of UDP-glucose. Gluconeogenesis – largely the reverse of glycolysis – converts mol-
ecules, such as lactate and amino acids, to glucose. Finally, the pentose phosphate shunt
serves the dual purpose of providing five-carbon sugars and generating NADPH; it bears a
strong similarity to the Calvin cycle of photosynthesis (Chapter 12).
13.1 GLYCOGEN METABOLISM
Glycogen represents a cell’s readily available energy store. These glucose polymers can have
molecular weights of up to 100 million. Most of the glycogen macromolecule has “straight-
chain” links (α1→4 linked glucosyl units), with about tenfold fewer “branch points” (α1→6
linkages). Thus, most glycogen metabolism involves adding or removing the α1→4 linked glu-
cosyl units at the non-reducing end. The other features of its metabolism involve the branch
points. At the core of the glycogen particle is a protein molecule called glycogenin. We begin
by describing the formation and breakdown of these immense molecules, and then examine
the regulation of glycogen metabolism as a model for controlling processes in cells.
Glycogen breakdown begins with the splitting of α1→4 links and, subsequently, the α1→6
branch points. While glycogen is more extensively branched than the amylopectin chains of
starch, the outer linear strands of glycogen still account for about half of its glucosyl residues.
Glycogen is found mainly in two tissues: liver and muscle. Liver glycogen is a glucose
buffer for feeding–fasting cycles. The liver synthesizes glycogen during the fed state, and
glucose units released to the blood during the fasted state to help maintain blood glucose
concentration. Glucose can also be released from liver glycogen in times of stress, such as
escaping from a predator. The glycogen broken down in muscle can only be used for energy
in the muscle cell itself because free glucose is not released. Thus, rapid muscle contraction
stimulates the immediate breakdown and utilization of glucosyl units from glycogen. Other
tissues have glycogen in far smaller amounts, and their function is similar to that of muscle:
glucose is not released; glucosyl units in the form of glucose-6-P is further metabolized.
With this broad background established, we turn to an examination of the separate pro-
cesses of glycogen synthesis and glycogen breakdown.
13.1.1 GLYCOGEN SYNTHESIS
The incorporation of glucose-6-P into glycogen proceeds by the pathway outlined in

Figure 13.2. The first step is the formation of glucose-1-phosphate (glucose-1-P), catalyzed
by phosphoglucomutase. This near-equilibrium enzyme has the same mechanism as the
289
290 13.1 Glycogen Metabolism
FIGURE 13.1 Carbohydrate pathways. The synthesis of glycogen (1) and its breakdown (2) are
connected to glycolysis through glucose-6-P, as is the catabolism of galactose (3). Also stemming
from glucose-6-P is the pentose phosphate shunt (4), which provides two molecules for the cell:
the pentose phosphate ribose-5-P and NADPH. Gluconeogenesis (5) is the synthesis of glucose
from certain precursors, such as the lactate illustrated here.
phosphoglyceromutase of glycolysis; that is, the phosphate groups are exchanged through
the intermediate formation of histidyl-phosphoryl groups on the enzyme.
In the next step, UTP reacts with G1P to form UDP-glucose, or activated glucose, cata-
lyzed by a synthetase. The other reaction product is pyrophosphate (PPi), hydrolyzed in an
ensuing reaction catalyzed by pyrophosphatase. Keeping a low [PPi] renders the UTP glucose
synthetase metabolically irreversible.
The synthetase reaction is a direct nucleophilic substitution: one of the oxygen anions of
the phosphate of glucose-1-P attacks the α-phosphoryl of UTP (Figure 13.3). Like the acetyl
portion of acetyl–CoA, the glucosyl portion of UDP-glucose is connected to a good leaving
group, making the incorporation of glucose units into glycogen energetically favorable.
The key regulated step of glycogen synthesis is catalyzed by glycogen synthase (Figure 13.4).
The overall reaction is shown in Figure 13.4a, in which all hydroxyl groups are displayed, with
the nucleophilic hydroxyl at the non-reducing end in red and the new incoming sugar ring
in green. In the abbreviated view of Figure 13.4b, the non-participating hydroxyl groups are
omitted. Intermediate steps of the mechanism are drawn in Figure 13.4c. Displacement of
the phosphate bond of UDP-glucose produces a glucose carbocation that is subsequently
attacked by the 4′-OH group of the non-reducing end of glycogen. A key piece of evidence
supporting this mechanism is inhibition of the enzyme by gluconolactone; its structure,
alongside the glucose carbocation, is shown in Figure 13.4d. The right-hand portions of both
molecules are flat, whereas the left-hand portions are identical, explaining why gluconolac-
tone binds to the active site of glycogen synthase. Both the inhibitor (lactone) and the inter-
mediate (carbocation) assume a six-membered ring structure in a half-chair configuration.
After about 10 glucosyl units have been added to the non-reducing end, a reaction cata-
lyzed by glycogen branching enzyme (Figure 13.5) removes a string of at least six glucosyl
units from the reducing end to an interior 6-hydroxy position. The creation of a new interior
α1→6 bond is known as a branch point.
Complete de novo synthesis of the glycogen molecule requires a core protein called gly-
cogenin, which serves as both an enzyme and a scaffold (Figure 13.6). Glycogenin is a dimer
Chapter 13 – Carbohydrate Pathways Related to Glycolysis 291
FIGURE 13.2 Glycogen synthesis. A mutase catalyzes the formation of glucose-1-P, followed
by the activation step, the formation of UDP-glucose. The key regulated step in the pathway is
catalyzed by glycogen synthase, which adds the glucose portion of UDP-glucose to an existing
chain of glucose residues.
containing two active sites terminating in acceptor tyrosine residues arranged head to toe.
UDP-glucose donates glucose residues first to the tyrosine hydroxyls. Next, a small chain
of glucosyl residues are incorporated. Subsequent buildup of the polymer involves glycogen
synthase and the branching enzyme, as above. A complete glycogen molecule is depicted in
Figure 13.7. Most glycogen synthesis, however, involves the incorporation of glucosyl units
onto an existing glycogen molecule; the glycogen molecule rarely breaks down completely.
We consider this breakdown process next.
13.1.2 GLYCOGENOLYSIS
Most glycogen breakdown is accomplished by a single reaction: the removal of α1→4

bound glucose residues from the non-reducing end. This reaction is catalyzed by glycogen
phosphorylase:
(13.1) ( glucose )n + Pi ® ( glucose )n -1 + glucose-1-P

The enzyme uses the bound cofactor pyridoxal phosphate (Chapter 7). The aldehyde
of the cofactor forms a Schiff base with a lysine residue of the enzyme; the phosphate
FIGURE 13.3 Formation of UDP-glucose.
FIGURE 13.4 Glycogen synthase mechanism. (a) Reaction structures. (b) Abbreviated reaction structures. (c) The mechanism
involves electron rearrangement from a lone pair of electrons on the ring oxygen and release of UDP to form the carbocation
intermediate, which is subsequently attacked by the 4 hydroxyl of the non-reducing end of glycogen. (d) The intermediate glu-
cose carbocation and the enzyme inhibitor gluconolactone are both forced into a half-chair configuration.
FIGURE 13.5 Branching enzyme reaction
FIGURE 13.6 Glycogenin. Glycogenin dimers are the scaffold for initiating the glycogen chain
as well as the catalysts for the incorporation of the first glucosyl units.
FIGURE 13.7 The glycogen particle (courtesy of Mikhael Haggerström).
group serves as an acid–base catalyst. While pyridoxal phosphate is a prosthetic group in

many other reactions, most of the coenzyme in the body is localized to muscle glycogen
phosphorylase.
The enzyme mechanism (Figure 13.8) involves the same glucose carbocation intermedi-
ate as glycogen synthetase. Accordingly, gluconolactone (Figure 13.4d) also inhibits glycogen
FIGURE 13.8 Mechanism of glycogen phosphorylase. Pyridoxal phosphate serves to posi-

tion the Pi substrate and participates in acid–base catalysis. Note the Pi attacks the carbocation
intermediate.
phosphorylase activity. The carbocation is formed after protonation and electron rearrange-
ment at the glucosyl unit of the non-reducing end of glycogen, leading to cleavage of the
α1→4 bond. Inorganic phosphate then attaches itself (as a nucleophile) to the carbocation to
form the product, glucose-1-P. The enzyme-bound pyridoxal phosphate forms an ionic bond
to the inorganic phosphate substrate, alternately deprotonating and protonating it.
Glycogen phosphorylase can progressively remove non-reducing end glycosyl units until
it stalls at about four residues from an α1→6 bond. At that point, debranching enzyme cata-
lyzes the removal of all but one glucosyl subunit from the branch, adding those glycosyl
units to the non-reducing end (Figure 13.9a). The remaining α1→6 bound glucosyl unit is
also removed by glycogen debranching enzyme. This glucosidase activity of the debranch-
ing enzyme occurs at a separate catalytic site from the activity that catalyzes relocation of
glycosyl residues to the non-reducing end. Glycogen branching enzyme is another example
of an enzyme with two different active sites, just like PFK-2 (a kinase/phosphatase) and the
mutase/phosphatase enzyme of the red blood cell shunt pathway of glycolysis (Chapter 9).
Overall, glycogen breakdown produces mostly glucose-1-P, with a small amount of free
glucose reflecting the number of branch points. In muscle, essentially no glucose leaves the
cell, as there is no glucose phosphatase activity. However, in the liver, glucose formation can
be the major fate of glycogen breakdown, an important activity to maintain blood glucose
concentrations.
Most of the well-established genetic defects due to deranged glycogen metabolism involve
enzymes of glycogen synthesis or degradation, with few exceptions (Box 13.1). Normal
(a)
(b)
FIGURE 13.9 Glycogen debranching enzyme reactions. (a) About four glucosyl units are
relocated from a branch point to a non-reducing end, leaving a single α1→6 bound unit. (b)
Glucosidase activity of debranching enzyme produces free glucose.
Box 13.1 Glycogen Storage Diseases
The glycogen storage diseases are a classic set of inborn errors of metabolism – genetic
defects related to glycogen metabolism. Because these are well-established disease
states, the missing gene is known in each case, and the consequences are understood.
Some of the defects are not direct deficiencies of glycogen enzymes themselves, but
rather in enzymes of connected pathways, such as glycolysis and gluconeogenesis.
The original classification used Roman numerals, assigned in order of their discovery.
Major storage diseases are listed in Table B13.1.
Consider type IV and type V in detail. Type IV, the loss of branching enzyme, leaves the
glycogen polymer unbranched and much less soluble. Because the branching enzyme is
more abundant in liver than in muscle, its deficiency is more obvious in the liver, which
becomes oversized (hepatomegaly) and scarred (cirrhotic). In type V, the deficiency in
muscle phosphorylase makes many types of exercise difficult because the glycogen
cannot be readily broken down as an energy source. Anaerobic exercise is particularly
affected (e.g., fast muscle). The more sustained type of aerobic exercise is supported by
lipid metabolism.
TABLE B13.1 Glycogen Storage Diseases

Type Deficiency Pathway Lesion
I Glucose-Pase (Ia) Gluconeogenesis, Glycogenolysis (liver)
Glucose-6-P Transporter (Ib)
II Lysosomal glucosidase Lysosomal catabolism
III Glycogen Debranching Enzyme Glycogen synthesis
IV Glycogen Branching Enzyme Glycogenolysis (muscle)
V Glycogen Phosphorylase Glycogenolysis (liver)
VI Phosphofructokinase Glycolysis (muscle)
control of glycogen metabolism ultimately comes down to the regulation of glycogen syn-
thase and glycogen phosphorylase. We next consider this issue in physiological situations.
13.1.3 PHYSIOLOGICAL CONTEXT OF GLYCOGEN METABOLISM
Glycogen serves as a readily available energy source for cells. As such, its metabolism must be
controlled in the face of varying physiological circumstances. While at least trace amounts
of glycogen are found in many animal cells, muscle and liver account for virtually all of the
body’s store of this polymer. Hence, we will consider the role of glycogen in just these two
tissues.
Muscle (in particular, fast or glycolytic muscle) uses glycogen as a fuel to support its
contraction. A rise in cytosolic Ca2+ triggers both muscle contraction and glycogenolysis.
Ca2+ stored in the muscle endoplasmic reticulum is released to the cytosol in response to
nerve-directed depolarization of the muscle’s plasma membrane. Contraction of the muscle
involves the movement of two protein complexes relative to each other: actin and myosin.
Myosin utilizes the bond energy of ATP to drive this process, during which ADP and Pi are
released. This “ATPase” accounts for most of the energy utilization in muscle contraction. It
is thus appropriate that the rise in Ca2+ concentration also serves to activate glycogen break-
down to support ATP formation.
Synthesis of glycogen is under the control of insulin, the hormone that is secreted into the
bloodstream during feeding conditions. Due to the large amount of skeletal muscle in the
body, skeletal muscle uptake of glucose and its incorporation into glycogen represents the
major depository of glucose after feeding.
Liver serves as a glucose buffer for the entire body, releasing to or taking up glucose from
the blood to maintain a relatively constant level. Breakdown of liver glycogen provides free
glucose to the blood for use by all of the cells in the body. The drop in blood glucose during
fasting conditions elicits a rise in glucagon, a hormone that activates liver glycogenolysis.
Liver also can release glucose from glycogen during conditions that elevate cytosolic Ca2+,
such as the adrenal glands’ release of epinephrine during a stress response. As in muscle,
insulin stimulates the synthesis of liver glycogen in the fed state.
In the following sections, we will examine hormonal regulatory systems of glycogen
metabolism imposed by insulin, in the muscle, and by glucagon and epinephrine in the
liver. Next, we will examine gluconeogenesis, the synthesis of glucose from non-carbohy-
drate precursors such as lactate. Finally, we will construct a broader picture of carbohy-
drate metabolism in the physiological contexts of the feeding–fasting and resting–exercise
transitions.
13.1.4 REGULATION OF GLYCOGEN METABOLISM BY GLUCAGON
Blood glucose concentrations drop routinely between meals, which signals the α-cells of the
pancreas to release glucagon. In the liver – its primary target – glucagon leads to the forma-
tion of a cytosolic messenger cyclic AMP (cAMP) by the process outlined in Figure 13.10.
Three proteins are involved: two membrane-bound proteins – the glucagon receptor and
the enzyme adenylate cyclase – and a G-protein, a membrane surface protein named for its
attachment to a guanine nucleotide. G-proteins bind either GDP or GTP. Like cytochrome c
of mitochondria (Chapter 11), the G-protein is mobile along the membrane surface.
When glucagon binds its receptor, it imposes a conformational change in the receptor
protein that enables it to bind the GDP:G-protein form. The receptor then catalyzes the
exchange of cellular GTP for its bound GDP. The GTP:G-protein dissociates from the gluca-
gon receptor, slides along the surface of the membrane, subsequently binding and activating
adenylate cyclase. The reaction catalyzed by this enzyme is:

(13.2) ATP ® cAMP + PPi
FIGURE 13.10 Glucagon and the G-protein. (a) G-protein (Gprot) shuttles between the gluca-
gon receptor (GR) and the effector, adenylate cyclase (AC). Gprot becomes activated when GR is
occupied by glucagon, enabling Gprot to exchange its bound GDP for GTP. The Gprot:GTP species
is the active form, stimulating AC, which catalyzes cAMP formation. (b) Gprot has an endogenous
enzymatic activity that hydrolyzes bound GTP constantly, releasing P; and leaving bound GDP
(inactive form). The Gprot:GTP form can be regenerated under the conditions of (a) above.
PPi is rapidly hydrolyzed (as in UDP-glucose formation in Section 13.1.1, GlycogenSynthesis),

ensuring metabolic irreversibility. As long as the G-protein has GTP bound, it can continue
to activate adenylate cyclase and drive the production of cAMP. However, G-protein has an
enzymatic activity of its own: a GTPase, which catalyzes:

(13.3) GTP:G-protein ® GDP:G-protein + Pi
The GDP-bound form of the G-protein can no longer activate adenylate cyclase, so this rep-
resents signal termination. However, as long as glucagon is bound to its receptor, GDP:G-
protein can slide along the membrane surface, engage the receptor, and undergo the exchange
reaction to the active, GTP-bound form. This system ensures that the effector – adenylate
cyclase – is activated only when the hormone is bound to the receptor.
Over 20 distinct G-proteins are known that transduce signals in the manner illustrated in
Figure 13.10, in many cases using an effector target other than adenylate cyclase. At least one
type of cancer is linked to a defect in the GTPase activity of a G-protein. The ras oncogene
results from an inactive GTPase, which leads to a state of constant activation and hyperactive
cell replication.
Overall, the result of glucagon action is an increase in cAMP concentration in the cytosol.
cAMP is a signal molecule that acts by binding to protein kinase A (PKA), an enzyme com-
posed of regulatory and catalytic subunits (Figure 13.11). In the resting (inactive) state, PKA
is a tetramer. When the concentration of cAMP is elevated, it binds the regulatory subunits,
releasing active catalytic monomers and dimeric regulatory subunits.
The reaction pictured in Figure 13.11 is an equilibrium. Thus, PKA can revert to an inac-
tive form if cAMP concentration is lowered and dissociates from the regulatory subunits.
Subsequently, regulatory and catalytic subunits combine, thereby inactivating PKA. This
inactivation will occur as glucagon levels drop and the G-protein reverts to its inactive form.
In addition, the constant presence of the enzyme phosphodiesterase, removes cAMP by cata-
lyzing its hydrolysis:

(13.4) cAMP + H 2O ® AMP
This provides another level of signal termination in addition to the GTPase activity of the
G-proteins.
As depicted in Figure 13.12, active PKA catalyzes the phosphorylation of two proteins
in glycogen metabolism. First, glycogen synthase (GS) is converted to an inactive form,
FIGURE 13.11 Activation of protein kinase A. Elevated concentrations of cAMP bind the regu-
latory subunit (R) of protein kinase A (PKA) and release it from the catalytic subunit (C), which is
the active form of PKA.
FIGURE 13.12 Protein kinase A regulation of glycogen metabolism. Protein kinase A (PKA)
leads to the inactivation of glycogen synthase (GS) and the activation of glycogen phosphory-
lase (GP), by converting both enzymes into their phosphorylated forms. PKA directly catalyzes
GS phosphorylation; however, GP is not a substrate for PKA. Rather, an intermediate kinase, GP
kinase, is a substrate for PKA. Once GP kinase is phosphorylated, this enzyme catalyzes the phos-
phorylation of GP.
reducing the rate of glycogen formation. Second, the enzyme glycogen phosphorylase
kinase is converted to its active form. Glycogen phosphorylase kinase acts on glycogen phos-
phorylase itself, catalyzing its phosphorylation and activation, leading to increased glycogen
breakdown. Thus, the effect on glycogen breakdown is indirect, involving an intermediate
enzyme phosphorylation. As we will appreciate in the next section, this provides a separate
opportunity for regulatory control.
13.1.5 REGULATION OF GLYCOGEN METABOLISM BY EPINEPHRINE
Whereas glucagon always leads to an increased concentration of cAMP, epinephrine pro-

duces different responses in different target tissues. The epinephrine receptor can activate
distinct plasma membrane-embedded receptor proteins. In muscle, epinephrine binds to
β-adrenergic receptors coupled to similar G-proteins of the type we have just considered and
leads to an increased cAMP and PKA activation.
However, in the liver of some species – including humans – epinephrine binds to the
α-adrenergic receptor, which binds a separate G-protein. This G-protein activates a distinct
effector system: the enzyme phospholipase C. Phospholipase C catalyzes the hydrolysis of
a phosphatidylinositol-4,5-P2 in the inner leaflet of the plasma membrane via the reaction
(Figure 13.13):

(13.5) PIP2 ® IP3 + DAG
Both products of this reaction are regulatory molecules. Inositol trisphosphate (IP3) is a
water-soluble molecule that binds a protein on the endoplasmic reticulum membrane sur-
face. Diacylglycerol (DAG) remains in the lipid phase of the membrane bilayer, activating a
protein at the cytosolic surface.
FIGURE 13.13 Phospholipase C and the formation of IP3 and DAG. The phospholipid phos-
phatidylinositol 4,5-bisphosphate – PIP2 – is a substrate for phospholipase C, the reaction shown
here. One product, diacylglycerol (DAG) remains in the membrane; the other, inositol triphos-
phate (IP3), is released to the cytosol.
FIGURE 13.14 Action of protein kinase C. Protein kinase C (PKC) is activated by the lipid-bound
modulator diacylglycerol (DAG). PKC associates with the inner plasma membrane. Once active,
PKC catalyzes the phosphorylation of its targets, such as the membrane-bound epinephrine
receptor (EpiR). The phosphorylated EpiR is inactive.
IP3 binds to and activates a calcium channel in the endoplasmic reticulum, allowing Ca2+
to move from the lumen of the endoplasmic reticulum to the cytoplasm. One consequence of
a rise in cytosolic Ca2+ concentration is the activation of glycogen phosphorylase kinase. This
enzyme is activated by Ca2+ regardless of its phosphorylation status. Phosphorylase kinase in
turn activates glycogen phosphorylase, so the latter enzyme is indirectly stimulated by the
rise in Ca2+. Ca2+ does not affect glycogen synthase, so it selectively activates glycogen break-
down in response to a rise in epinephrine.
Epinephrine can also produce a rise in [DAG] through the activation of phospholipase C,
although most DAG responsible for cell signaling is derived from other phospholipid precur-
sors, such as phosphatidylcholine. Moreover, the rise in DAG is slower than the increase in
Ca2+ concentration. In liver, for example, an increase in cytosolic Ca2+ concentration occurs
in less than one minute.It takes at least 10 times as long for an appreciable rise in DAG con-
centration. DAG is an activator of protein kinase C (PKC), which has multiple targets in the
cell. One of these is the epinephrine receptor itself (Figure 13.14), where it serves to dampen
receptor response to epinephrine binding. In this way, epinephrine binding to its receptor
eventually leads to inactivation of that receptor, a phenomenon known as down regulation.
While these two mechanisms provide a complete picture of the regulation of glycogen
metabolism by epinephrine, the actions of this hormone are somewhat broader. The mecha-
nism depends upon the subtype of the epinephrine receptor and includes one extra intracel-
lular target: a potassium channel (Box 13.2).
13.1.6 REGULATION OF GLYCOGEN METABOLISM BY INSULIN
A rise in blood glucose level after a meal triggers the release of insulin from the pancreatic
β-cells. This 50-amino acid peptide hormone is an anabolic signal. In both muscle and liver,
insulin leads to increased glycogen synthesis. The most prominent and well-studied effect is
on muscle tissue.
Insulin stimulation of glycogen stores ultimately leads to the activation of glycogen syn-
thase. A key proximal regulator of glycogen synthase is the enzyme glycogen synthase
kinase 3 (GSK3), named for a region of phosphorylation sites on glycogen synthase. An
overview is shown in Figure 13.15. Upon insulin binding its receptor in the extracellular
space, intracellular events initiated by a conformational change in the insulin receptor ulti-
mately lead to the phosphorylation and inactivation of GSK3. This lifts the inhibition that the
dephosphorylated form of GSK3 exerts on glycogen synthase, activating glycogen synthase.
A more detailed view of insulin action is presented in Figure 13.16. Step (1) shows insulin
binding the exterior portion of the insulin receptor. In step (2), the bound insulin recep-
tor undergoes a conformational change in its intracellular part and activates the intrinsic
kinase activity of the insulin receptor. The insulin receptor catalyzes the phosphorylation
Box 13.2 Epinephrine Receptor Subtypes and Associated Mechanisms
Our description of the different intracellular mechanisms in response to an elevation in

epinephrine is centered on glycogen metabolism. This emphasis is appropriate in that
the origins of our understanding of many intracellular regulatory mechanisms were stud-
ies of glycogen metabolism. Two major mechanisms – one involving cAMP and the other
Ca2+ – affect the synthesis and breakdown of glycogen. However, a more comprehensive
understanding of epinephrine receptors – more widely known as adrenergic receptors
based on the alternative name adrenaline – requires that we move beyond glycogen
metabolism.
Historically, the first division of adrenergic receptors was between the α and β types.
The α-adrenergic receptors were believed to act through a Ca2+-dependent mechanism,
whereas the β–adrenergic receptors act through a cAMP-dependent mechanism. Later
it was discovered that there are subtypes of these receptors. It happens that all known
β-subtypes (e.g., β1, β2, β3) act through a cAMP mechanism. However, there are two
major subtypes of the α-adrenergic receptor, and they have distinct mechanisms. The
α1-subtype involves Ca2+ as described. However, the α2-subtype does not involve Ca2+ or
cAMP.
The α2-adrenergic receptor activates a potassium channel in excitable cells such as
nerve and muscle. As a result, K+ leaves the cell, and it becomes more polarized, or less
excited. This action is not unique to the α2-adrenergic channel; the same intracellular
mechanism holds for acetyl-choline in the heart cell. Having the same mechanism for
different receptors is also the case for cAMP elevation, elicited both by glucagon and the
β-adrenergic receptor. In the nervous system, the activation of α2-adrenergic receptors is
elicited not by epinephrine by but norepinephrine, a neurotransmitter structurally and
functionally related to epinephrine:
OH OH
H
HO N HO NH2
CH3
HO HO
Epinephrine Norepinephrine
Substances mimicking the action of norepinephrine in the central nervous system (ago-
nists) can suppress the activity of certain neurons that trigger a pain response, as they
raise the firing threshold.
of tyrosine residues on its own cytosolic domains. The phosphorylated insulin receptor next
catalyzes the phosphorylation of insulin receptor substrate 1 (IRS-1), specifically at tyro-
sine residues; this is step (3). Thus, the insulin receptor plays three roles in this signaling
pathway: as hormone receptor in step 1; a self-phosphorylating activity acting as a kinase and
substrate in step 2; and as a kinase acting on the external substrate IRS-1 in step 3.
The phosphorylated tyrosine residues of IRS-1 can bind to proteins that contain
Src homology-2 (SH2) binding domains (Figure 13.17). This is step (4) of Figure 13.16.
Phosphatidylinositol phosphate 3-kinase (PI3K) is an enzyme that has a regulatory subunit
with SH2 binding domains. When PI3K binds phosphorylated IRS-1, it becomes activated.
PI3K catalyzes the phosphorylation of a membrane lipid in step (5):
Phosphatidylinositol-P2 ( PIP2 ) + ATP

(13.6)
® Phosphatidylinositol-P3 ( PIP3 ) + ADP
FIGURE 13.15 Overview of insulin stimulation of glycogen synthase. Insulin binds the insulin
receptor (IR), followed by a series of intermediate steps, ultimately leading to the phosphoryla-
tion and inactivation of glycogen synthase kinase 3 (GSK), thereby lifting the inhibition of glyco-
gen synthase (GS).
FIGURE 13.16 The insulin mechanism. When insulin binds the insulin receptor (1), the latter
becomes a tyrosine kinase (2), phosphorylating itself as well as the insulin receptor substrate
(IRS-1) (3). Phosphorylated IRS-1 binds targets such as the phosphatidylinositol 3-kinase (Pl3K)
(4), which catalyzes the formation of phosphatidyl 3,4,5-trisphosphate (PIP3) (5). PIP3 serves as a
binding site for phospholipid dependent kinase (PDK) (6), which catalyzes the phosphoryla-
tion and activation of protein kinase B (PKB) (7). PKB catalyzes phosphorylation and inactivation
of glycogen synthase kinase 3 (GSK) (8). This phosphorylation lifts GSK inhibition of glycogen
synthase, leaving it less phosphorylated, and activated.
FIGURE 13.17 The SH2 domain. The ribbon diagram shows the coil-sheet-coil pocket struc-
ture of this domain, with the phosphotyrosine residue binding to the pocket. (Source: Lee, C.H.,
Kominos, D., Jacques, S., Margolis, B., Schlessinger, J., Shoelson, S.E., Kuriyan, J. 1994. Crystal struc-
tures of peptide complexes of the amino-terminal SH2 domain of the Syp tyrosine phosphatase.
Structure 2:423–438. Protein Data Base #1AYA; prepared by Molly Steinbach.)
As shown in Figure 13.16, the inositol head group of the lipid product PIP3 has three phos-
phates that serve to anchor the next protein, phospholipid-dependent kinase (PDK), which
is step (6). In step (7), PDK catalyzes the phosphorylation and activation of protein kinase B
(PKB). PKB, in turn, catalyzes the phosphorylation of GSK3 in step (8). This phosphorylation
inactivates GSK3, leaving glycogen synthase less phosphorylated, and hence with greater
activity. The result is an increase in glycogen synthesis.
The insulin receptor is one member of a class of receptor tyrosine kinases, which also
includes the insulin-like growth factor 1 (the growth hormone mediator) and epidermal
growth factor. All have the same pattern. First, a hormone binds the receptor at the cell
exterior, leading to a conformation change in the receptor protein that activates its intrinsic
kinase activity. Next, there is a phosphorylation of one or more intracellular proteins. The
phosphorylated proteins serve as binding sites for yet other signaling protein intermediates.
The intracellular steps transmit the signal to specific targets, dictated by the extracellular
hormonal milieu. Protein kinase C catalyzes the phosphorylation and inactivation of the
insulin receptor, in a similar manner to the epinephrine receptor (Figure 13.14).
13.1.7 REGULATION OF GLYCOGEN METABOLISM BY AMP KINASE
AMP-dependent protein kinase (AMPK) is an enzyme found in virtually all cells. Because
the activator AMP is elevated under periods of energy deprivation, this type of control is dis-
tinct from hormonal regulation, which involves cell-to-cell signaling. The generation of AMP
requires the breakdown of cellular ATP to ADP, followed by the action of adenylate kinase:
(13.7) ADP + ADP AMP + ATP
We first encountered the adenylate kinase in Chapter 8, as a reaction serving to convert AMP
to ADP. As it is a near-equilibrium reaction, it can also proceed in the direction written in
Equation 13.7, when ADP is elevated. For example, during intense muscle contraction, ATP
utilization outstrips ADP phosphorylation. As another example, limiting oxygen availability
304 13.2 Gluconeogenesis
FIGURE 13.18 AMP kinase control of glycogen metabolism. AMP Kinase (AMPK) is activated
allosterically by AMP, and by phosphorylation of the enzyme by the upstream kinase, AMP Kinase
Kinase (AMPKK). These regulators interact; AMP makes AMPK a better substrate for AMPKK. AMPK
has two opposing effects on glycogen synthetase. It catalyzes direct phosphorylation of GS to
an inactive phosphorylated form (acting as a GSK-2). However, AMPK also actives glucose uptake
by stimulating GLUT4, leading to an increased [G6P], an allosteric activator of GS. In some cases,
the latter is more influential, and AMPK leads to glycogen accumulation.
(anoxia) can also lead to the accumulation of ADP. AMPK itself is the substrate of a protein
kinase; in fact, phosphorylation of AMPK is essential for its activation (Figure 13.18). Binding
AMP to AMPK makes it a better substrate for this upstream kinase. Once activated, AMPK
phosphorylates glycogen synthase as a glycogen synthase kinase 2 (GSK-2), distinct from
GSK-3 in insulin signaling. Like GSK-3, AMPK also inactivates glycogen synthase. However,
an opposing action of AMPK is the stimulation of glucose uptake by muscle cells, which
elevates the concentration of glucose-6-P, an activator of glycogen synthase. The net result of
these opposing actions on glycogen synthase is stimulation, as AMPK activation is known to
cause the accumulation of glycogen.
13.2 GLUCONEOGENESIS
The pathway for forming glucose from non-carbohydrate precursors occurs almost exclu-
sively in the liver. The kidney has the complete pathway, but contributes little to total body
blood glucose. Gluconeogenesis is also found in non-mammalian species, such as yeast and
some plants (Box 13.3).
Lactate, glycerol, and many amino acids (such as alanine) are converted to glucose in the
mammalian liver. These precursors take separate pathways initially, but they ultimately con-
verge. We will consider the steps from lactate to glucose in detail here. The prime importance
of this pathway is to maintain blood glucose, as glycogen (the first line of supply) is depleted.
Most of the gluconeogenic reactions are reversals of the near-equilibrium steps of gly-
colysis. Only the metabolically irreversible steps – pyruvate to phosphoenolpyruvate (PEP),
fructose-1,6-P2 to fructose-6-P, and glucose-6-P to glucose – involve specific gluconeogenic
enzymes. We start with the first reaction of the pathway, catalyzed by lactate dehydrogenase.
13.2.1 LACTATE DEHYDROGENASE AS A GLUCONEOGENIC ENZYME
Lactate dehydrogenase catalyzes the last step of glycolysis and the first step in gluconeogenesis:

(13.8) Lactate + NAD+ ® Pyruvate + NADH
Box 13.3 Word Origins: What’s Really New about Gluconeogenesis?
Two parts of the term gluconeogenesis indicate that it is new: both the direct Latin neo
and the concept of beginnings in genesis. Genesis also could be viewed as generated,
but linked with neo, it strongly conveys the meaning of origins. But is it really new?
The Cori cycle is a combination of two pathways that span different tissues in the
body. Glucose, once ingested, is stored mainly in liver and muscle. Not only that, its
metabolism by these tissues is quantitatively of paramount significance. Muscle takes up
most of the dietary glucose in the body by virtue of its mass, and uses it to support con-
traction. Some of the carbon is completely oxidized, and some converted by muscle to
blood lactate and carried to the liver. In the Cori cycle, carbon is shuttled between these
two organs, as suggested in Figure B13.1
Thus, gluconeogenesis could be viewed as the regeneration of glucose carbon that
was converted to lactate by the muscle in a continuing cycle. This regeneration led bio-
chemist Mitchell Halperin to suggest the pathway be renamed glucopaleogenesis.
FIGURE B13.1 The Cori cycle.
In the gluconeogenic direction, the standard free energy change is extremely unfavorable,
with ΔG0 = +46 kJ/mol. However, this has no metabolic significance. Under cellular condi-
tions, the reaction is near-equilibrium, like most gluconeogenic steps. Hence, LDH is not
subject to external regulation, but only by changes in substrate and product concentrations.
Like lactate DH, the other near-equilibrium steps in gluconeogenesis use the same
enzymes as in glycolysis. Their direction in gluconeogenesis is the reverse of glycolysis. Our
focus in gluconeogenesis will be on the reactions required to enable flow in the reverse direc-
tion to the metabolically irreversible steps of glycolysis. This first is the conversion of pyru-
vate to PEP.
13.2.2 PYRUVATE TO PEP
The conversion of pyruvate to PEP is the rate-limiting stage of gluconeogenesis. The reverse
process in glycolysis requires just one enzyme (pyruvate kinase). However, gluconeogenesis
requires two enzymes, the expenditure of two high-energy phosphates, and the shuttling of
substrates between the cytosol and mitochondria.
The first metabolically irreversible enzyme of gluconeogenesis is pyruvate carboxylase,
which exists exclusively in the mitochondrial matrix. Cytosolic pyruvate is transported into
the mitochondria, using the pyruvate transport system (the same transporter used in pyru-
vate entry to the mitochondria for oxidation, Chapter 10). The overall reaction catalyzed by
pyruvate carboxylase is:

(13.9) Pyruvate + CO2 + ATP ® Oxaloacetate + ADP + Pi
The incorporation of CO2 requires the bound cofactor biotin (Chapter 7), which is covalently
bound to the enzyme through an amide linkage with a lysine residue.
The mechanism shares one feature with the pyruvate dehydrogenase complex, in that a
“swinging arm” moves a molecular fragment between active sites. In pyruvate carboxylase,
there are just two active sites, residing in separate portions of the same enzyme. A biotin-
lysine arm becomes carboxylated at one site, then moves to another, where the carboxyl
group is added to pyruvate (Figure 13.19a). In the first site, HCO3− is attached to biotin in a
reaction assisted by ATP:
(13.10) HCO3 - + ATP + Enz-biotin ® Enz-biotin-CO2 + ADP + Pi
The mechanism is shown in Figure 13.19b. CO2 (as its hydrate, bicarbonate) reacts with ATP
to form a carboxyphosphate intermediate. One of the nitrogens of biotin acts as a nucleo-
phile, attacking the carboxyphosphate and forming a carboxy-biotinylated enzyme.
Once the swinging arm moves the carboxyl-biotin portion to the second active site, the
sequence shown in Figure 13.19c takes place. A series of concerted reactions between car-
boxybiotin and pyruvate produces oxaloacetate and the enol form of biotin. Biotin is in equi-
librium with this tautomer and can be redeployed for another cycle of catalysis at the first
active site of pyruvate carboxylase.
Phosphoenolpyruvate carboxykinase (PEPCK) is the next step, catalyzing the reaction:

(13.11) Oxaloacetate + GTP ® PEP + GDP + CO2
The electron rearrangements that decarboxylate and phosphorylate the oxaloacetate are
illustrated in Figure 13.20. The two substrates are brought in close proximity on the enzyme
with the assistance of a Mn2+ ion that attracts the negative charges of the carboxylate of oxa-
loacetate and the phosphate of GTP.
While there are no known allosteric modulators of PEPCK, this enzyme is widely con-
sidered the rate-limiting step of gluconeogenesis under various physiological conditions. Its
activity is regulated by genetic control of PEPCK protein production rather than the minute-
to-minute modulation of enzyme activity, such as those described for glycogen metabolism.
In brief, glucagon leads to increased PEPCK protein content, whereas insulin leads to dimin-
ished PEPCK protein content, thereby setting the pace of gluconeogenesis.
Note that the CO2 incorporated into pyruvate carboxylase is released by PEPCK. Hence,
there is no net incorporation of CO2 in gluconeogenesis, in contrast to the Calvin cycle of
plants. When pyruvate carboxylase was first discovered, this distinction between net incor-
poration (plants) and using CO2 as a temporary inclusion (animals) was unknown, which
made the discovery controversial (Box 13.4).
13.2.3 INDIRECT TRANSPORT OF OXALOACETATE FROM MITOCHONDRIA TO CYTOSOL
The overall conversion of pyruvate to PEP requires a carboxylation and subsequent decar-
boxylation, using the reactions of (13.10) and (13.11). The carbon fate is:

(13.12) pyruvate ® oxaloacetate ® PEP
As we have described, the production of oxaloacetate is catalyzed in the mitochondria by

pyruvate carboxylase. However, a substantial fraction of PEPCK, which catalyzes the sub-
sequent conversion of oxaloacetate to PEP, occurs in the cytosol. No transporter exists to
FIGURE 13.19 Mechanism of pyruvate carboxylase. (a) Overall mechanism: The biotin arm
of the enzyme reacts with ATP and HCO3− at one site, attaching CO2 to the biotin and then
moves the other, where carboxylation of pyruvate occurs. (b) Carboxylation of biotin: ATP and
HCO3− react to form carboxyphosphate, which is attacked by a nitrogen of the biotin to produce
carboxybiotin. (c) Pyruvate carboxylation: A series of concerted electron rearrangements shown
attach CO2 to pyruvate, producing oxaloacetate and the enol form of the attached biotin. This
enol-biotin tautomer is in equilibrium with the original cofactor form. Once biotin is restored to
its original form, the enzyme can engage in another cycle of catalysis.
FIGURE 13.20 Mechanism of PEP carboxykinase. The decarboxylation of oxaloacetate is

linked to phosphorylation. Oxaloacetate and GTP are positioned on the enzyme with the assis-
tance of Mn2+, enabling electron rearrangement to form the products.
permit oxaloacetate to cross the inner mitochondrial membrane. Instead, oxaloacetate is

indirectly transported by conversion to other molecules that do have transporters in the
inner mitochondrial membrane. The two different routes are possible:
1. Reduction to malate.
2. Transamination to aspartate.
Both malate and aspartate have selective transport proteins in the inner mitochondrial mem-
brane. Thus, they act as surrogates of oxaloacetate. After malate or aspartate cross the inner
mitochondrial membrane and enter the cytosol, they are converted back to oxaloacetate. The
reactions use separate mitochondrial and cytosolic isozymes of the relevant enzymes.
Malate DH is present in mitochondria (also serving as part of the Krebs cycle):

(13.13) Oxaloacetate + NADH ® Malate + NAD+
The malate is then transported to the cytosol, where a separate malate DH catalyzes the
reverse reaction:

(13.14) Malate + NAD+ ® Oxaloacetate + NADH
Both malate DH enzymes catalyze near-equilibrium reactions. The direction of the reaction
is dictated by the relative concentrations of substrates and products established by the pro-
duction of oxaloacetate (by pyruvate carboxylase in the mitochondria) and by its removal (by
PEPCK in the cytosol).
Alternatively, oxaloacetate can be converted to aspartate in a mitochondrial reaction cat-
alyzed by aspartate aminotransferase:

(13.15) Oxaloacetate + Glutamate ® Aspartate + a-Ketoglutarate
Aspartate crosses the inner mitochondrial membrane on its own transporter and is the sub-
strate of the cytosolic enzyme, aspartate aminotransferase:
Box 13.4 CO2 Fixation in Animals?
The incorporation of CO2 in the pyruvate carboxylase reaction was first discovered
in the early 1930s when isotopically labeled carbon became available to researchers.
Incorporation of CO2 into stable products in animal tissue was met with skepticism by the
scientific community. Even then it was well understood that plants incorporate CO2 as the
building block of the organism, but animals do not. The reaction to the pyruvate carbox-
ylation findings was expressed by the response to the key publication by Harland Wood
and Chester Werkman: it was lampooned as the “wouldn’t work” reaction. Only later did
the pattern emerge in animal metabolism: whenever CO2 is incorporated in a reaction
such as pyruvate carboxylase, it is followed by a corresponding decarboxylation, such
as PEPCK. The purpose is to energetically drive the latter reaction, because the escape of
CO2 to the gas phase contributes to making reactions exergonic.

(13.16) Aspartate + a-Ketoglutarate ® Oxaloacetate + Glutamate
Like the malate DH isozymes, the aspartate transaminases are also near-equilibrium.
The pathways for oxaloacetate transfer are illustrated in Figure 13.21. Note that, with
malate as the intermediate, NADH is effectively transported from mitochondria to cytosol.
However, lactate DH also generates NADH, and this is re-oxidized later at glyceraldehyde-P
DH: the reverse of the glycolytic sequence. Hence, gluconeogenesis from lactate uses aspar-
tate as the intermediate, because the use of malate would cause an overproduction of NADH.
More oxidized precursors for gluconeogenesis, such as pyruvate itself, use malate as an inter-
mediate. The availability of two separate transport systems thus provides metabolic flexibil-
ity to meet the redox state needs for gluconeogenesis from different substrates.
13.2.4 FRUCTOSE-1,6-P2 TO FRUCTOSE-6-P
The bypass of the phosphofructokinase step is catalyzed by fructose-1,6-P2 phosphatase. The

reaction is:

(13.17) Fructose-1, 6-P2 ® Fructose-6-P + Pi
and it is metabolically irreversible. The mechanism is a typical hydrolysis, in which a

nucleophilic attack by water on the 1-phosphate bond of the substrate releases inorganic
phosphate (Pi).
The enzyme is allosterically activated by citrate and inhibited by fructose-2,6-P2 . Both
of these modulators have opposing effects on phosphofructokinase, for which citrate is
an inhibitor and fructose-2,6-P2 is an activator. Together these two enzymes constitute a
substrate cycle, historically described as a futile cycle because the net result is simply to
split ATP:
(13.18) PFK : ATP + Fructose-6-P ® ADP + Fructose-1, 6-P2

(13.19) FBPase: Fructose-1, 6-P2 ® Fructose-6-P + Pi
__________________________________________________________
Net reaction : ATP ® ADP + Pi
While it is true that the simultaneous operation of such opposing reactions does lead to a
net loss of ATP energy, the advantage is that control of either gluconeogenesis or glycolysis
becomes more sensitive when both directions are subject to regulatory modulation.
FIGURE 13.21 Two shuttles for mitochondrial oxaloacetate. In the mitochondrial matrix, oxa-
loacetate forms either malate (MDH) or aspartate (ATA). Both malate and aspartate, unlike oxalo-
acetate, have exchangers in the mitochondrial inner membrane, allowing them to appear in the
cytosol. Isoenzymes of MDH or ATA catalyze oxaloacetate formation in the cytosol.
13.2.5 GLUCOSE-6-P TO GLUCOSE
The conversion of glucose-6-P to glucose is catalyzed by the enzyme glucose-6-phos-

phatase, a combination of a transport system and a catalytic phosphatase, involving the
endoplasmic reticulum. Glucose-6-P is first transported into the lumen of the endoplasmic
reticulum. Subsequently, a relatively nonspecific phosphatase (Pase in Figure 13.22) catalyzes
the hydrolysis to glucose. The other product is Pi, which enters the cytosol using an exchange
transport protein. The transporter carries glucose-6-P into the lumen of the ER as it carries
Pi into the cytosol. Finally, GLUT7 is a specific transporter for the entry of glucose into the
cytosol. Glucose can exit the cell using the same GLUT2 plasma membrane transporter used
for hepatic glucose uptake.
Regulation of the glucose-6-Pase system is exerted through increased protein formation
(i.e., by control of genetic expression) of the catalytic subunit during starvation, directed by
glucagon, and through its suppression in the fed state, directed by insulin. Together with
glucokinase, glucose-6-Pase forms the following substrate cycle:

(13.20) GK: Glucose + ATP ® Glucose-6-P + ADP

(13.21) G6Pase: Glucose-6-P ® Glucose + Pi
__________________________________________________
Net reaction: ATP ® ADP + Pi
In addition to the two substrate cycles already described, the reactions between PEP and
pyruvate constitutes a third substrate cycle, if we ignore the nucleotide specificities involved
(i.e., we substitute NTP for either ATP or GTP):

(13.22) PK: PEP + NDP ® Pyruvate + NTP
FIGURE 13.22 Glucose-6-Pase. A transporter enables G6P entry to the endoplasmic reticu-
lum (ER), where a membrane-bound phosphatase (Pase) catalyzes glucose formation. A separate
transporter, GLUT7, moves glucose back into the cytosol.

(13.23) PC : Pyruvate + NTP ® Oxaloacetate + NDP + Pi

(13.24) PEPCK: Oxaloacetate + NTP ® PEP + NDP
______________________________________________________
Net reaction: NTP ® NDP + Pi
Regulation of any step of this cycle can regulate gluconeogenesis. Thus, short-term control
(activation) of gluconeogenesis can be exerted through PKA-dependent phosphorylation and
the inactivation of pyruvate kinase. All three substrate cycles are shown in Figure 13.23.
13.2.6 PATHWAY INTEGRATION: GLYCOLYSIS, GLYCOGEN

METABOLISM, AND GLUCONEOGENESIS
We can gain biochemical insight into physiological processes by examining pathways integra-
tion. Here we examine two common events: the feeding–fasting transition and the resting–
exercise transition. We will confine our discussion to the principal routes of carbohydrate
metabolism: glycolysis, gluconeogenesis, and glycogen metabolism.
13.2.6.1 THE FEEDING–FASTING TRANSITION
During a meal, glucose moves from the digestive tract to the bloodstream, rising there to
a peak of about 12 mM. The elevated blood glucose concentration triggers the pancreas to
release insulin, which in turn activates glycogen synthesis in the liver and muscle. Elevated
insulin activates glycolysis in liver and adipose tissue, and suppresses liver gluconeogenesis.
Glycogenolysis is depressed by insulin in both the liver and muscle. In muscle, insulin stimu-
lation of glucose transport is critical for the incorporation of the sugar into glycogen. These
actions, summarized in Figure 13.24, counteract the rise in blood glucose. The carbohydrate
pathways prominent in the fed state are glycogen synthesis and glycolysis.
After a meal (the postprandial state), glucose concentration drops, suppressing insulin
release from the pancreatic β-cells, and stimulating glucagon release from the pancreatic
α-cells. This activates glycogen breakdown and gluconeogenesis in the liver, and the inhi-
bition of glycolysis, releasing glucose to the blood. The decrease in insulin concentration
reduces glucose uptake by muscle. The adrenal gland releases epinephrine in response to
a low blood glucose concentration. This signaling originates in the central nervous system,
carried to the periphery via nerve fibers that synapse on the adrenal glands. Epinephrine
FIGURE 13.23 Substrate cycles. Three substrate cycles catalyze net hydrolysis of ATP (or NTP)
using the metabolically irreversible reactions of gluconeogenesis and glycolysis. This provides
fine control of both pathways.
stimulates glycogen breakdown in the liver. The glucose phosphate formed in the cell can-
not enter glycolysis because this pathway is suppressed under fasting conditions; glucose is
released to the blood. The three hormones discussed are summarized in Table 13.1.
13.2.6.2 THE RESTING–EXERCISE TRANSITION
The skeletal muscle, by virtue of its mass, is a major consumer of glucose even in the resting state.
During exercise, skeletal muscle can increase its energy utilization by 100-fold. Accordingly,
dramatic events in metabolic pathways are needed to support this increased energy utilization.
The regulation of muscle metabolism is largely intrinsic; the increased AMP that
results from the extensive adenine nucleotide turnover activates AMPK, drives glucose
uptake, and stimulates glycolysis. As discussed above, AMPK can lead to overall stimula-
tion of glycogen deposition, as it stimulates glucose uptake by muscle and increases [G6P],
an allosteric activator of glycogen synthase. However, AMPK also directly phosphorylates
and inhibits glycogen synthase, so that the effect on glycogen synthesis may depend upon
the overall physiological state. During exercise, blood flow is shunted away from the liver
and toward muscle, diminishing the metabolic role of the liver. In the post-exercise state,
blood flow returns to the liver, and excess lactate accumulated by muscle glycolysis is
converted to glucose by liver gluconeogenesis. An extreme example is found in the alliga-
tor (Box 13.5).
A further effect of exercise is both intrinsic and hormonal: a rise in the concentration of
intracellular Ca2+. Epinephrine increases cytosolic Ca2+ concentration, which stimulates the
breakdown of liver glycogen. In muscle, Ca2+ concentration is elevated during the contrac-
tion cycle itself, which also activates glycogen breakdown.
FIGURE 13.24 Regulation of carbohydrate pathways.
TABLE 13.1 Key Hormones of

Physiological Glycogen Regulation
Hormone Origin Target
Insulin Pancreatic β-cell Liver, muscle, fat
Glucagon Pancreatic α-cell Liver
Epinephrine Adrenal gland Liver, muscle, fat
Two other pathways connected to glycolysis proceed at much lower rates: the pentose
phosphate shunt and the more specialized route of galactose metabolism.
13.3 THE PENTOSE PHOSPHATE SHUNT
Like glycolysis, the pentose phosphate shunt occurs in all cells in the body. This pathway has
two distinct contributions to the cell. First, it partially oxidizes glucose-6-P in a reaction
that also reduces NADP+ to NADPH. These pyrimidine nucleotides are virtually identical
to NAD+ and NADH, apart from an extra phosphate group located far from the nicotin-
amide ring. The standard redox potential of the NAD+/NADH couple is identical to that of
the NADP+/NADPH couple. In the cell, however, the NADP+/NADPH couple is far more
reduced – by a factor of up to five orders of magnitude. The distinction of the extra phosphate
group between these cofactor pairs enables binding discrimination between different dehy-
drogenase enzymes. The NADPH functions as an electron donor for biosynthetic reactions
and as a reductant for suppressing oxidative damage.
314 13.3 The Pentose Phosphate Shunt
Box 13.5 Alligators and the Resting–Exercise Transition
The rapid and powerful muscle contraction of the alligator jaw muscles while attacking
prey can be sustained for only several minutes. It is an extreme example of glycolysis sup-
plied by glycogen breakdown in white muscle (fast or glycolytic muscle). This anaerobic
metabolic response characterizes the similar brief, yet powerful, muscle movements of
the weight-lifter.
In the alligator, lactate produced by muscle glycolysis can be converted to glucose
by the liver and then transported to muscle for glycogen synthesis (see the Cori cycle,
Figure B13.1). However, hepatic gluconeogenesis is relatively slow, and the long resting
period of the alligator reflects this restoration process. A similar pattern applies to the
closely related species, the crocodile. Ogden Nash made one distinction in the poem The
Purist: a field biologist is told that an alligator ate his bride. The scientist cannot suppress
a smile; he points out it was, in fact, a crocodile. Crocodiles are indeed much larger than
alligators and more likely to attack humans. The biologist would also presumably know
the habitat of each species; crocodiles are more widely distributed around the globe. A
further distinction between these crocodilians is noted for nitrogen metabolism in the
Chapter 15 Addendum.
The other function of the pentose phosphate shunt is the production of different sugar
phosphates, most notably ribose phosphates used in the synthesis of nucleotides. The end-
products of the pentose phosphate shunt are intermediates in glycolysis. Cells can readily
control the relative amount of pentose or NADPH using a simple equilibrium adjustment,
which we will elaborate once we have examined the overall pathway.
13.3.1 OXIDATIVE STAGE
All of the reactions of the oxidative stage of the pentose phosphate shunt are metabolically
irreversible. The overall process is:

(13.25) Glucose-6-P + 2 NADP+ ® Ribulose-5-P + CO2 + 2 NADPH
Glucose-6-P DH catalyzes the first step of the oxidative stage (Figure 13.25). The enzyme is
allosterically inhibited by NADPH. Its mechanism (hydride transfer) is similar to other dehy-
drogenases (e.g., lactate DH). The product is the cyclic ester 6-phosphogluconolactone, which
is hydrolyzed to the open chain 6-P-gluconate in a lactonase-catalyzed reaction. A second
NADPH is produced in the ensuing 6-P-gluconate DH reaction. The mechanism is another
example of oxidative decarboxylation, just like the mechanisms of the isocitrate DH reaction
in the Krebs cycle and the malic enzyme in lipid metabolism.
13.3.2 NONOXIDATIVE STAGE
All of the enzymes in the nonoxidative stage of the pentose phosphate shunt catalyze near-
equilibrium reactions. This portion of the pentose cycle strongly resembles the Calvin cycle
of Chapter 12. We can understand the flow of carbon in the nonoxidative portion of the pen-
tose phosphate shunt by imagining the Calvin cycle operating in reverse, with some modi-
fications. We will visualize this pathway in three separate ways. First, we will consider the
reactions individually. Next, we will view the pathway sequence and determine the carbon
balance. Finally, we will examine a pseudo three-dimensional diagram of the pathway and
compare it to the Calvin cycle.
FIGURE 13.25 Pentose phosphate shunt: oxidative reactions. Two dehydrogenases – reactions (1) and (3) – produce NADPH.
Hydrolysis of the lactone product of reaction (2) is catalyzed by a lactonase. All of these reactions are metabolically irreversible.
13.3.2.1 REACTIONS OF THE NONOXIDATIVE STAGE
Five enzymatic reactions convert ribulose-5-P (Ru5P) – the endpoint of the oxidative stage –
into the glycolytic intermediates GAP and F6P. Two of these convert Ru5P into different
five-carbon sugar phosphates, as illustrated in Figure 13.26. An epimerase catalyzes the for-
mation of xylulose-5-P (Xu5P). An isomerase catalyzes the formation of ribose-5-P (R5P).
Some of the R5P is diverted into the formation of ribonucleotides (see Chapter 15).
The remaining three reactions of this stage are of two types: transketolase and trans-
aldolase. These enzymes catalyze reactions that remove a portion of one substrate and add
it to another. The cleavage pattern of the two enzymes is illustrated in Figure 13.27. In the
transketolase reaction, cleavage occurs between the carbonyl and the α-carbon. In the trans-
aldolase reaction, the sugar-phosphate is split between the carbonyl and the β-carbon. In
each case, the upper fragment joins the aldehyde group of an acceptor. We have already come
across the transketolase mechanism in our study of the Calvin cycle. The transaldolase reac-
tion is unique to this pathway. The mechanism for the enzyme is similar to aldolase (Chapter
8). The first part of both the aldolase and the transaldolase mechanisms is the same up to
the cleavage step. In aldolase, the enzyme-bound fragment is released by hydrolysis, whereas
in transaldolase, the enzyme-bound fragment condenses with a new aldehyde (the second
substrate), forming a new aldol product.
The last three reactions of the nonoxidative stage are presented in Figure 13.28. Adjacent
dots indicate carbon fates in both substrate and product. In the first reaction (Figure 13.28a),
two 5-carbon sugar phosphates undergo a transketolase reaction, producing GAP and S7P.
These products, in turn, become the substrates for the transaldolase-catalyzed reaction, pro-
ducing E4P and F6P (Figure 13.28b). Finally, a second molecule of Xu5P reacts with E4P in
another transketolase-catalyzed reaction to produce GAP and F6P (Figure 13.28c).
The pathway flow from the five-carbon sugar phosphates is made explicit in Figure 13.29.
The inputs are outlined in red; the products in blue. Overall the reaction stoichiometry is:

(13.26) 3 Ru5P ® 2 F6P + 1 GAP
which indicates a balance of 15 carbons (3 five-carbon inputs) converted to 15 carbons (2

6–carbon and one 3–carbon outputs). As both F6P and GAP are intermediates in glycolysis,
and the pentose phosphate pathway is initiated by the first intermediate of glycolysis, it is
theoretically possible to return all of the carbon – apart from CO2 – back to the pathway.
Putting this together with the oxidative arm, the overall balance is:

(13.27) 6 G6P ® 6CO2 + 6 Ru5P ® 4 F6P + 2 GAP
316 13.3 The Pentose Phosphate Shunt
FIGURE 13.26 C5 lnterconversions in the pentose phosphate shunt. Ribulose-5P (Ru5P) is

converted to the epimer xylulose-5-P (Xu5P) and to the isomer ribose-5-P (R5P), catalyzed by
epimerase and isomerase, respectively. These are the first steps in the nonoxidative portion of
the pentose phosphate shunt.
FIGURE 13.27 Cleavage points of transketolase and transaldolase catalyzed reactions. The
points of cleavage of each of these reactions are indicated. One is α to the carbonyl; the other is
β to the carbonyl. In each case, the acceptor molecule is an aldehyde.
13.3.2.2 A PSEUDO THREE-DIMENSIONAL VIEW AND COMPARISON TO THE CALVIN CYCLE
A pseudo three-dimensional view of the nonoxidative segment of the pentose phosphate

cycle is presented in Figure 13.30a . In the adjacent panel are the similar reactions of the
Calvin cycle (Figure 13.30b). For both pathways, the intermediates at the vertices of the cube
are identical.
For the pentose phosphate shunt, Ru5P carbon enters at Xu5P (epimerase) and at R5P
(isomerase). These two undergo a transketolase reaction, and their products undergo a trans-
aldolase reaction (the side faces of the cube). Finally, the top face shows E4P (produced in the
transaldolase reaction and highlighted in red) reacting with XSP in a second transketolase
reaction, producing the products GAP and F6P that enter glycolysis.
FIGURE 13.28 Transaldolase and transketolase reactions of the pentose phosphate shunt.
Dots indicate the carbon fates of each reaction.
For the Calvin cycle segment, GAP is the sole input, and X5P and R5P are the out-
puts. As indicated in Figure 13.30b, reactions involving DHAP (isomerase and aldol-
ases) are required to generate three other intermediates that participate in transketolase
reactions. The appearance of DHAP in the center of the bottom right square makes
this pathway distinct. Note that the Calvin cycle does not have a transaldolase reac-
tion. It also requires an extra phosphatase step (not shown) to convert the SBP to S7P.
Otherwise, the reactions directly correspond to the pentose phosphate shunt but in the
reverse direction.
Of the two pathways, the pentose phosphate shunt is more broadly distributed biologi-
cally, occurring in both plants and animals. It also displays metabolic flexibility: the oxidative
and nonoxidative stages proceed at different rates depending upon the metabolic needs for
NADPH or ribose. We next consider how this is accomplished.
318 13.4 Galactose Utilization
FIGURE 13.29 Pathway of the nonoxidative pentose phosphate shunt.
13.3.3 DISTRIBUTION BETWEEN NADPH PRODUCTION AND RIBOSE-5-P
Cellular demand fluctuates between requirements for NADPH (e.g., for biosynthetic path-
ways such as lipid synthesis) and ribose-5-P (e.g., for nucleotide synthesis). The relative
needs can be accommodated due to the near-equilibrium nature of the nonoxidative seg-
ment of the pentose phosphate pathway. If demand for ribose carbon increases, more will
be drawn from the pathway, in which case less will be returned to glycolytic intermediates.
If relatively more NADPH is needed, then more carbon can be returned to glycolytic inter-
mediates, and less ribose-5-P is removed from this segment of the pathway. This simple
means of endogenous control underscores the self-regulating nature of near-equilibrium
reactions.
13.4 GALACTOSE UTILIZATION
Galactose is derived from lactose, the disaccharide found in milk. After hydrolysis of lactose
to glucose and galactose, catalyzed by the intestinal enzyme lactase, the galactose is uniquely
metabolized by the liver through a pathway intersecting with the glycogen metabolism as
both involve UDP-glucose.
Galactose catabolism is outlined in Figure 13.31. First, galactose is converted to galac-
tose-1-P, catalyzed by the liver-specific galactokinase (analogous to fructose metabolism
in the liver). In the next step, the galactose moiety exchanges with the glucose moiety of
UDP-glucose, catalyzed by uridylyltransferase. The products of this reaction are glucose-
1-P and UDP-galactose. Finally, an epimerase converts UDP-galactose to UDP-glucose,
recycling the substrate for the uridylyltransferase. As a result, the pathway produces net
glucose-1-P and catalytic amounts of UDP sugars. Notice that the pathway is not com-
plete in a single pass, but requires an initial formation of UDP-glucose; the glucose-1-P
is formed only in a second pass. This is yet another example of repetition–variation in
metabolic pathways.
The mechanism of the UDP-galactose epimerase, like other epimerase catalyzed
reactions such as the conversion of Ru5P to Xu5P (Figure 13.26) involves a symmet-
rical intermediate, a feature common to isomerization reactions. As illustrated in
FIGURE 13.30 Three-dimensional pathway views of the pentose phosphate shunt and the
Calvin cycle. (a) The nonoxidative reactions of the pentose phosphate shunt. The transaldolase
reaction is indicated with red arrows. (b) The analogous Calvin cycle reactions run in the opposite
direction, with no transaldolase reaction. Aldolase conversions involving the intermediate dihy-
droxyacetone P (DHAP) occur instead.
Figure 13.32a, the conversion can be described overall as inverting the stereochemistry

of the hydroxyl group at C4. The mechanism is outlined in Figure 13.32b. The enzyme
uses NAD+ to remove a hydride from the hydroxyl group, forming a ketone intermediate.
The ketone is the symmetrical intermediate. In the other half of the mechanism, NADH
binds and adds hydride to the opposing face, forming the stereochemically distinct alco-
hol. Thus, while this enzyme utilizes the hydride transfer coenzyme NADH, it is not a
redox reaction.
While galactose metabolism is a specialized route, UDP-glucose can be otherwise
modified, for example by oxidation of the C6 hydroxyl group to the acid, forming UDP-
glucuronide. This is activated glucuronide serves as a sugar phosphate donor in drug
conjugation reactions in the liver, in which foreign substances in the body (xenobiotics)
become more polar. The increased water solubility ensures xenobiotic excretion by the
kidney.
320 Summary
FIGURE 13.31 Galactose catabolism. Galactose phosphorylation is catalyzed by liver galacto-

kinase, after which a uridylyltransferase catalyzes the exchange of sugars on the UDP carrier. An
epimerase catalyzes interconversion of UDP-galactose with UDP-glucose.
FIGURE 13.32 Mechanism of epimerase. The mechanism requires the redox nucleotide pair
NAD+/NADH, for the purpose of creating the symmetrical intermediate ketone. Overall, the redox
state is balanced.
SUMMARY
Pathways connected to glycolysis include glycogen synthesis, glycogen breakdown, gluco-

neogenesis, the pentose phosphate shunt, and galactose utilization. Most of the glycogen in
mammals (and, consequently, most of its metabolism) is found in liver and muscle. The syn-
thesis of glycogen involves two key enzymatic steps: conversion of UDP-glucose to glycogen,
catalyzed by glycogen synthase, and the formation of branches, catalyzed by the branching
enzyme. De novo synthesis of the glycogen molecule, a rare event, involves the small protein
glycogenin, both as scaffold and enzyme for the first few glucosyl units. The key enzyme
of glycogenolysis is glycogen phosphorylase, which catalyzes the removal of glucosyl units
from the non-reducing end of the polymer to form glucose-1-P. The debranching enzyme
catalyzes the relocation of chains from the α1→6 branch points to the non-reducing ends of
glycogen, leaving a single glucosyl unit in an α1→6 linkage. A glucosidase activity of the deb-
ranching enzyme removes this unit to form free glucose. Several control systems target two
critical enzymes of glycogen metabolism: glycogen synthase and glycogen phosphorylase.
In liver, glucagon leads to protein kinase A activation and phosphorylation (and inactiva-
tion) of glycogen synthase, and phosphorylation (and activation) of glycogen phosphorylase
kinase, which in turn phosphorylates (and activates) glycogen phosphorylase. Epinephrine
can increase intracellular Ca2+, activating both glycogen phosphorylase kinase and glyco-
gen phosphorylase activity. Insulin, through a receptor tyrosine kinase, leads to a series of
binding and phosphorylation steps that inactivate glycogen synthase kinase, which lifts a
tonic inhibition of glycogen synthase. A rise in AMP, triggered by increased ATP utilization,
activates the AMP kinase, which phosphorylates and inactivates glycogen synthase. AMPK
can paradoxically also activate glycogen synthesis as it can stimulate glucose transport. The
overall effect depends on the physiological state. These regulatory systems appear widely
in metabolic pathways, including gluconeogenesis. That pathway is essentially glycolysis in
reverse, with three unique segments that are metabolically irreversible. First, pyruvate is
converted to PEP using transport steps that link mitochondrial pyruvate carboxylase (pyru-
vate → oxaloacetate) to cytosolic PEP carboxykinase (oxaloacetate → PEP). Second, fruc-
tose-1,6-P2 is converted to fructose-6-P, catalyzed by fructose bisphosphatase. Finally, the
glucose-6-Pase system, which involves transport between the cytosol and the endoplasmic
reticulum, converts glucose-6-P to glucose. All three of these segments constitute metabolic
cycles, in which both glycolytic and gluconeogenic enzymes are simultaneously active. Each
involves a net loss of a high-energy phosphate, but enables more exquisite metabolic control,
as every enzyme of the cycle can serve as a control point. The pentose phosphate shunt is
a side branch of glycolysis from glucose-6-P. One portion of this pathway – the oxidative
arm – produces NADPH and pentose phosphates. The NADPH is used in reductive synthesis
reactions. The pentose phosphates, primarily ribose-5-P, can be used for nucleotide synthesis
or converted through reactions similar to those of the Calvin cycle to glycolytic intermedi-
ates. The assimilation of galactose, derived from the milk sugar lactose, is a liver pathway
that converts galactose to UDP-galactose and then to UDP-glucose for entry into glycolysis.
REVIEW QUESTIONS
1. Prior to certain physical exercise, such as sprinting, a nutritionist recommends a diet

heavy in carbohydrates. What is the rationale?
2. Explain why a transaldolase enzyme is necessary for the pentose phosphate shunt,
but not in the Calvin cycle.
3. Which control systems are active during muscle contraction? During starvation?
4. How does insulin lead to the activation of glycogen synthase? Is there an insulin effect
on glycogen phosphorylase?
5. Early studies with isotopic labeling showed that muscle could produce glucose, which
raised the possibility that muscle undergoes gluconeogenesis. What was the reason
behind this conclusion?
6. How is the relative formation of pentose phosphate shunt products NADPH and
ribose-P regulated?
7. What steps of glycogen metabolism are inhibited by gluconolactone?
CHAPTER 13 ADDENDUM: PHOSPHORYLATION OF GLYCOGEN
Microscopic visualization of cells (including electron microscopy) shows groups of spherical

masses suggesting clusters of glycogen molecules attached to cellular actin. Attached to the
322 Key Terms
external surface of the glycogen molecules are the enzymes that synthesize and degrade the
glycogen chain.
An unusual and still not well understood structural feature of glycogen is the presence of
phosphate groups attached to the free hydroxyls (e.g., 2,3, or 6 positions) in glycogen. This
exists to the extent of about 1 phosphate per 1000 glucosyl residues. One suggestion for the
presence of the phosphate groups is the ability to unfold the branches of glycogen.
While the presence of phosphate was earlier shown in plant starch (amylopectin), its pres-
ence in glycogen was established by a disorder known as Lafora disease, in which glycogen
has abnormal branches. Clinically, Lafora disease is associated with epilepsy, although the
link between glycogen metabolism and epilepsy is unknown. One cause of the disorder is the
mutation of genes encoding laforin, a carbohydrate phosphatase that catalyzes hydrolysis
of phosphate groups from glycogen. A separate cause of the disease is a defect in malin, a
component of the ubiquitin pathway for protein breakdown (see Chapter 17).
Whereas the phosphatase for glycogen is established, the carbohydrate kinase remains
unknown. There are at least two such kinases known in photosynthetic organisms that
incorporate phosphate into amylopectin.
Thus, glycogen metabolism involves not only extensive phosphorylation and dephosphor-
ylation of enzymes regulating the synthesis and breakdown of glycogen but the incorporation
of phosphate into glycogen itself.
KEY TERMS
adrenergic receptors
Cori cycle
down regulation
glucagon
gluconeogenesis
glucose-6-phosphatase
glycogen branching enzyme
glycogen storage diseases
glycogen phosphorylase
glycogen phosphorylase kinase
glycogen synthase kinase
glycogenin
insulin
insulin receptor substrate-1
intermediary metabolism
Lafora disease
laforin
malin
phosphoenolpyruvate carboxykinase
phospholipid-dependent kinase
postprandial state
protein kinase C
pyruvate carboxylase
receptor tyrosine kinases
src homology-2 (SH2) binding domains
transaldolase
transketolase
xenobiotic
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Lipid Metabolism 14
A defining characteristic of animal life is the use of fat for energy rather than carbohydrates.
As an extreme example, migratory birds can travel a thousand miles on their lipid stores,
enough for more than a week of nonstop flight. Aside from energy, all cells require mem-
branes, so they need to synthesize phospholipids. In the present chapter, we examine the
pathways for utilizing lipid for energy and for synthesis of the major lipid molecules: fatty
acids, triacylglycerols, phospholipids, and cholesterol. In addition to their energy storage and
barrier roles, lipids also serve as important signal molecules, such as inositol lipids. We begin
our study of the major lipid pathways by examining the fate of dietary lipids.
14.1 ABSORPTION OF DIETARY LIPIDS
There are two routes of lipid assimilation in mammals: endogenous and exogenous. The
endogenous pathway is the biosynthesis of fatty acids from excess dietary carbohydrates and
protein. The exogenous pathway is dietary lipid.
Most dietary lipids are triacylglycerols; the remainder is mostly phospholipid (represent-
ing membrane material of ingested foodstuffs), cholesterol, and its derivatives. After food
enters the stomach, the organ’s churning action converts solid nutrients into a liquefied sus-
pension called chyme (Figure 14.1). Once chyme enters the duodenum (the first portion of
the small intestine), the lipid portion combines with bile, a mixture of bile salts, phospholip-
ids, and cholesterol. Bile is produced by the liver and enters the small intestine through the
bile duct. Along the way, bile also flows into a blind pouch, the gall bladder. This storage sac
can deliver a bolus of bile during the ingestion of a fatty meal. The mixture of bile with lipid
forms smaller lipid droplets as the bile salts coat the outside of the particles, a process called
emulsification.
The exocrine pancreas secretes several enzymes and proteins into the duodenum, includ-
ing pancreatic lipase and colipase. Colipase assists the binding of the lipase to the suspen-
sions of lipid particles. Lipase catalyzes the hydrolysis of the ester bonds in triacylglycerols,
releasing fatty acids. Fatty acids are passively transported (Box 14.1) across the plasma mem-
brane of the intestinal epithelia. Once inside these cells, triacylglycerols (and cholesterol
esters) are resynthesized (Figure 14.2). The cells also synthesize a protein called an apoli-
poprotein, which along with phospholipid forms the exterior of a spherical particle known
as a chylomicron. Chylomicrons leaving the intestinal epithelia enter the lymphatic system.
Lymphatic vessels terminate at the vena cava, at which point chylomicrons enter the blood-
stream. Once in the blood, the triacylglycerols in the chylomicrons are hydrolyzed back to
fatty acids and taken into adipocytes. The hydrolysis reaction is catalyzed by esterases cova-
lently attached to the walls of blood vessels near adipocytes. Fatty acids entering into the
adipocytes reform triacylglycerols. Adipocytes are thus the storage site for fatty acids in the
form of triacylglycerols.
325
326 14.2 Fatty Acid Oxidation
FIGURE 14.1 Lipid absorption: view from the intestine. (1) Stomach reduces food to a liquid
suspension called chyme. (2) Bile stored in the gall bladder enters the intestine, emulsifying the
fat. (3) Pancreas secretes lipase and colipase, which lead to (4) hydrolysis of triglycerides and
uptake of fatty acids by the intestinal epithelia.
Box 14.1 Fatty Acid-Binding Proteins
While fatty acid uptake into cells is described here as passive, there is extensive evidence
for many different proteins that may transport fatty acids across membranes and may
serve as intracellular binding proteins. Evidence in favor of this hypothesis comes mainly
from genetic knockout experiments or gene insertions and the subsequent observations
of the effects on fatty acid metabolism. While these proteins may influence transport
or storage, controversy remains as to whether this is their true function. For one thing,
the ability to bind a fatty acid is not a selective property of a protein. In fact, virtually all
globular proteins bind fatty acids. Even proteins with fatty acid-binding as their specific
role can be replaced, as in the case of a group of people found to have no serum albumin
and yet have no obvious clinical problems. In that case, a compensating increase in the
blood γ-globulin protein served to bind blood fatty acids.
In many cases, fatty acid transport across the plasma membrane is not saturable. Even
when it is, this analysis is complicated by binding fatty acids to proteins in the cell exterior
and the limited further metabolism of the fatty acid in the cell. Some fatty acid-binding
proteins have been found to have other functions. For example, the plasma membrane
protein CD36 is widely assigned the function of fatty acid transport protein. Yet, CD36
is also known as the thrombospondin receptor, as it binds the antiangiogenic serum
factor thrombospondin. Thus while it is possible that fatty acids use plasma membrane
proteins to cross the membrane, it appears that there is little selectivity to this process.
14.2 FATTY ACID OXIDATION
In the fed state, the blood concentration of free fatty acids is low (0.01 mM), but in the fasted
state, this value can rise 100-fold. As blood glucose falls, the autonomic nervous system trig-
gers epinephrine release from the adrenal glands. Epinephrine binds to receptors in the adi-
pocytes, which leads to the hydrolysis of stored triacylglycerols (and cholesterol esters), thus
Chapter 14 – Lipid Metabolism 327
FIGURE 14.2 Chylomicron synthesis in intestinal epithelia. Fatty acids are esterified in the epi-
thelia and form chylomicron particles after being coated with apolipprotein and phospholipids.
The chylomicron exits the endothelial cell into the extracellular space and is transported through
the lymphatic circulation until it enters the bloodstream.
forming free fatty acids through a stimulation of hormone-sensitive lipase (Figure 14.3). The
fatty acids, bound to the protein albumin in the blood, reach tissues for oxidation, such as
muscle, heart, and liver.
Friedrich Wohler, famous for the synthesis of urea in the laboratory (1828) and dispel-
ling organic mysticism, had observed even earlier (1824) that fatty acids could be completely
oxidized to carbon dioxide and water in animals. Hence, fatty acid oxidation is the earliest
pathway to be established. In an early form of metabolic labeling, Fritz Knoop (1904) fed
dogs fatty acids of various chain lengths having a phenyl group attached at the methyl end. A
pattern was clearly apparent from the urine products: carbon fragments are released in pairs
from the carboxyl end of the chains.
The pathway for fatty acid oxidation can be conceptually divided into three steps
(Figure 14.4): activation, the conversion of a fatty acid to an acyl-coenzyme A (acyl-CoA);
transport, importing acyl-CoA into the mitochondria; and β-oxidation, the conversion of
fatty acyl-CoA to acetyl-CoA. The activation step occurs in the cytosol, while β-oxidation
occurs within the mitochondrial matrix. The fatty acids are mixtures of saturated and unsat-
urated molecules and usually have 14, 16, or 18 carbons. We will present the pathway for the
oxidation of palmitate and briefly consider modifications needed to oxidize unsaturated fatty
acids and those with different chain lengths.
14.2.1 ACTIVATION
The activation step converts the fatty acid to the CoA ester:

(14.1) R -COO- + CoA-SH + ATP ® R-C ( = 0 ) SCoA + AMP + PPi
This reaction is catalyzed by fatty acyl-CoA synthase and is metabolically irreversible,

assisted by rapid pyrophosphate hydrolysis, and catalyzed by pyrophosphatase:
(14.2) PPi ® 2Pi

FIGURE 14.3 The adipocyte in fed and fasted states. In the fed state (top), the adipocyte
synthesizes some of the fatty acids from glucose; most arrive from chylomicrons (dietary fat) or
VLDL (endogenously synthesized fat). The fatty acid is stored as triacylglycerol. In the fasted state
(bottom), triacylglycerol is hydrolyzed in the adipocytes. While only the hormone-sensitive lipase
is indicated, there are two other lipases involved in the complete breakdown of triglycerides. The
released fatty acid travels in the blood bound to albumin and is delivered to muscle and liver for
oxidation.
Pyrophosphatase is also involved in uridine diphosphate (UDP)-glucose formation for gly-

cogen synthesis (Chapter 13). The mechanism of fatty acyl-CoA synthase is analogous to
asparagine synthetase. Here, the thiol of the CoA cofactor (explicit when written as CoA-SH)
serves as the nucleophile that displaces the acid phosphate intermediate.
14.2.2 TRANSPORT
Movement of fatty acyl-CoA into the mitochondria is indirect because no transport pro-
teins exist for CoA or its esters in the mitochondrial inner membrane. Like the shuttles for
FIGURE 14.4 General steps in fatty acid oxidation.
FIGURE 14.5 Carnitine acyl transferase I (CAT I).
NADH and oxaloacetate (Chapter 13), an indirect transport system is required to produce
mitochondrial acyl-CoA, involving the intermediate carnitine. First, fatty acyl-CoA is con-
verted to its carnitine ester in a reaction catalyzed by carnitine acyltransferase I, or CAT I
(Figure 14.5). The hydroxyl group of carnitine initiates an attack on the acyl-CoA carbonyl,
releasing CoA-SH and producing fatty acyl carnitine. Carnitine, named for its discovery in
meat (muscle tissue), is present in high concentration in muscle and liver. Animals both
synthesize carnitine and obtain it from the diet, so it is usually not limiting for fatty acid
oxidation.
In the next step of the carnitine shuttle, a mitochondrial transport protein catalyzes cyto-
solic acyl-carnitine exchange for mitochondrial carnitine. Once acyl-carnitine enters the
mitochondrial matrix, an isozyme of carnitine acyl transferase, CAT II, catalyzes the conver-
sion of fatty acyl-carnitine to fatty acyl-CoA. Thus, the CAT II reaction runs in the reverse
direction to CAT I (Figure 14.6). The carnitine released by CAT II is recycled by exchange
transport back to the cytosol, where it can participate in the CAT I reaction.
CAT I is the key regulatory step in the cellular process of fatty acid oxidation. The regula-
tor is malonyl-CoA, which allosterically inhibits CAT I. As we will see below, malonyl-CoA
is an intermediate of fatty acid synthesis in the liver, so there is a coordination of synthesis
and oxidation of fatty acids.
FIGURE 14.6 Import of fatty acids into mitochondria. An exchange protein catalyzes the
import of acyl-carnitine to the mitochondria and the export of carnitine to the cytosol. Carnitine
is the other product of the CAT II reaction.
β-OXIDATION
14.2.3
The mitochondrial fatty acid oxidation steps remove two carbons at a time from the chain of
the fatty acid, at a position β to the carbonyl. The sequence is known as β-oxidation, having
the overall reaction:

(14.3) ( Fatty acyl )n -CoA ® ( Fatty acyl )n -2 -CoA + Acetyl-CoA
The pathway is illustrated in Figure 14.7 in parallel with analogous steps of the Krebs cycle.
The Krebs cycle sequence from succinate to oxaloacetate involves the reduction of ubiqui-
none (UQ), a hydration, and NADH production. In the first step of β-oxidation, catalyzed
by fatty acyl-CoA dehydrogenase, electrons are removed from the two methylene carbons
adjacent to the carbonyl and transferred to the mobile cofactor UQ. The complex catalyzing
this transfer contains the fixed electron carriers FAD and Fe-S protein. This process is analo-
gous to the succinate dehydrogenase complex, which receives electrons from two adjacent
methylene carbons that are transferred first to FAD, then to an Fe-S protein, and finally to
UQ. Both enzyme complexes consist of integral membrane proteins and interact with the
mobile cofactor UQ.
A hydratase catalyzes the second reaction, the addition of water across a double bond,
with the same mechanism as fumarase. Both reactions are stereospecific, producing the
L-isomer exclusively. The hydroxyl group is subsequently oxidized to a ketone in a reaction
catalyzed by acylhydroxy dehydrogenase. The mechanism – removal of a hydride ion to form
NADH – is identical to the malate dehydrogenase reaction in the Krebs cycle.
The product of the preceding reactions, a ketoacyl-CoA, undergoes nucleophilic attack by
the sulfur of a CoA-SH molecule, catalyzed by a thiolase. This yields acetyl-CoA and a short-
ened (n-2) acyl-CoA. From this point, the newly shortened acyl-CoA repeats the four steps
just described. While the steps are repeated, different isozymes exist for the first enzyme
(acyl-CoA DH) with distinct specificities for long-, medium-, and short-chain acyl-CoA
molecules.
The mitochondrial oxidation of fatty acids is another example of repetition–variation,
somewhat different from the Krebs cycle, for example. In the Krebs cycle and galactose
catabolism, the pathway is incomplete until many passes occur that ultimately balance the
carbons. In fatty acid oxidation, the number of passes is set by the number of two-carbon
fragments produced, for example, 8 in the case of palmitate oxidation. A second distinction
is that the intermediates remain the same in the Krebs cycle, but the output requires many
passes for ultimate balance. In fatty acid oxidation, the intermediates are themselves the
source of variation.
FIGURE 14.7 Mitochondrial steps in fatty acid oxidation. The mitochondrial steps, known as
β-oxidation, are shown on the left side. Along the right side are similar reactions that occur in the
Krebs cycle. Only the last reaction, catalyzed by thiolase, is distinctive.
14.2.3.1 UNSATURATED FATTY ACIDS
Unsaturated fatty acids, such as oleate (Box 14.2), are abundant in mammalian blood and
can be oxidized by a slight modification of the pathway just described. After three rounds of
β–oxidation (Figure 14.8), an intermediate is formed with a cis-double bond beta to the car-
bonyl. This species is a substrate for an isomerase that catalyzes conversion to a trans-double
bond alpha to the carbonyl. The remaining oxidation steps proceed as before.
14.2.3.2 ODD CARBON-NUMBERED FATTY ACIDS
Odd-numbered fatty acids are rare in human serum, amounting to less than 1% of the
total. One source is cow milk, which contributes C15:0 fatty acids. Such fatty acids undergo
beta-oxidation normally until the final product, which is propionyl-CoA instead of acetyl
CoA. From this point, a different series of mitochondrial reactions ensue, as illustrated in
Figure 14.9. First, proprionyl-CoA is converted to S-methylmalonyl-CoA in a carboxylation
reaction catalyzed by proprionyl-CoA carboxylase. This biotin-dependent carboxylation
reaction has the same mechanism as pyruvate carboxylase (Chapter 13). A racemase enzyme
Box 14.2 Word Origins: Oleate
Oleate is derived from the Greek olea, meaning olive. Triacylglycerols of the olive flesh
(pericarp) consist mostly of oleic acid (18Δ9). The related term oleaginous is used as a
rough synonym for anything oily. Oleaceous is more strictly defined as being a mem-
ber of the family Oleaceae (of which Olea is a subcategory, a genus); these include lilac
and tropical trees. Surprisingly, the oleo part of oleomargarine has an unusual origin:
it was the name of the first margarine, produced in the 1860s from a beef fat extract
subjected to a churning process. Since the 1950s, margarine has been produced by the
partial chemical saturation of plant oils, including oleic acid, and several others, such as
peanut, soybean, and cottonseed oils. The high heat required for the process causes the
rearrangement of some cis double bonds to the trans configuration. There is currently a
health concern over the consumption of margarine because eating trans fats is correlated
with cardiovascular disease.
More recently, Proctor and Gamble produced the eponymous olestra as a fat substi-
tute. Chemically, olestra resembles triacylglycerols because it is a multiple fatty acid ester,
but the backbone is sucrose rather than glycerol. Not all hydroxyl groups of sucrose are
substituted in commercial preparations of olestra (Figure B14.2). Olestra is not broken
down in the intestine by esterases, and it is not incorporated as a dietary lipid, so it is
calorie-free. However, use of olestra is controversial, because many consumers suffered
intestinal disorders arising from undigested lipids passing through their intestinal tract.
An additional problem is that olestra decreases the absorption of fat-soluble vitamins.
Nonetheless, the enormous increase in the incidence of obesity and consequent diabe-
tes has spurred the development of this and, undoubtedly, many future new fat substi-
tutes for consumers.
FIGURE B14.2 Olestra.
catalyzes the conversion of S-methylmalonyl-CoA to the R-isomer. Finally, a complex mutase

reaction utilizing Vitamin B12 converts R-methylmalonyl-CoA to succinyl-CoA. Vitamin
B12, also known as cobalamin, is an elaborate cofactor reminiscent of a heme – but with
a cobalt ion chelating four imidazole nitrogens. The mechanism involves the intermediate
formation of a Co3+–carbon bond. In the liver, the succinyl-CoA can be converted to glucose;
FIGURE 14.8 Oleate oxidation. After three rounds of β-oxidation, oleate becomes a 3-cis fatty
acyl-CoA. The latter is a substrate for an isomerase that leads to the 2-trans fatty acyl-CoA that
can proceed through the remainder of the usual steps for fatty acid oxidation.
FIGURE 14.9 Unique steps in odd chain fatty acid oxidation. Following shortening the fatty
acid and acetyl-CoA production, the final 3-carbon intermediate is propionyl-CoA. This is carbox-
ylated in the propionyl CoA carboxylase reaction, stereochemically rearranged by a racemase,
and converted to succinyl-CoA through a vitamin B12-dependent mutase. Succinyl CoA is a Krebs
cycle intermediate, but not the substrate of the cycle; it enters the gluconeogenic pathway.
334 14.3 Ketone Body Metabolism
thus, odd chain number fatty acids are partially gluconeogenic. Usually, fatty acid oxidation
proceeds from even-numbered fatty acids, which produce acetyl-CoA exclusively, and can-
not be converted to glucose. Hence, the adage “you can’t convert fat into sugar”. While the
number of odd chain fatty acids is small, and the amount of carbon that forms succinyl-CoA
is also a minor portion of the molecule, this does constitute an exception to the rule.
14.2.3.3 SHORTER AND LONGER FATTY ACIDS
Fatty acid chains that are relatively short or very long have a different catabolic fate. Those
fatty acids having about 10 or fewer carbons (medium-chain fatty acids) bypass the cytosolic
activation step because they are not substrates for the cytosolic acylation reaction. These
shorter-chain fatty acids can diffuse directly into the mitochondrial matrix, and undergo
activation to CoA esters. Thus, medium and short-chain fatty acids are carnitine-indepen-
dent and escape malonyl-CoA control of their oxidation.
Very long-chain fatty acids, such as erucic acid (22Δ13), derived from canola oil, are par-
tially oxidized (a few rounds of β-oxidation) in the peroxisomes, a separate organelle from
the mitochondria. Peroxisomal oxidation reactions are not energy-linked; instead, elec-
trons are used to reduce O2 to H2O2, which is subsequently converted to H2O in a reaction
catalyzed by the namesake peroxidase enzymes of the peroxisome. The shorter fatty acids
produced are medium-chain fatty acids, activated within the mitochondria, and are not sub-
strates of CAT I. In this way, very long-chain fatty acids and shorter-chain fatty acids merge;
neither are subject to malonyl-CoA regulation.
14.3 KETONE BODY METABOLISM
Ketone bodies are produced exclusively in the liver as a spillover pathway during excess
production of acetyl-CoA, usually arising from fatty acid oxidation. Ketone bodies can also
be derived from the oxidation of certain amino acids, known as ketogenic amino acids
(Chapter 15). Typically, a rise in ketone bodies in the blood parallels an increase in blood
fatty acid concentration.
The first stage in ketone body synthesis is the formation of the acetyl-CoA carbanion
(Figure 14.10a). In the next step, the acetyl-CoA anion displaces CoA from a separate mol-
ecule of acetyl-CoA to form acetoacetyl-CoA (Figure 14.10b). In the subsequent 3-hydroxy-
3-methylglutaryl (HMG)-CoA synthase step, another acetyl-CoA carbanion attacks the
carbonyl of acetoacetyl-CoA, resulting in the tertiary alcohol, HMG-CoA. The mecha-
nism of these condensation reactions is similar to that of citrate synthase (Chapter 10). The
HMG-CoA degrades to acetyl-CoA and acetoacetate through a lyase-catalyzed reaction
(Figure 14.10c).
Acetoacetate, the first of the ketone bodies, reacts with NADH in a reaction catalyzed
by β-hydroxybutyrate dehydrogenase (a near-equilibrium enzyme), to form the product β–
hydroxybutyrate, the second ketone body (Figure 14.11). The liver cannot further metabolize
either acetoacetate or β-hydroxybutyrate, and these are both transported out of the cell.
Once in the bloodstream, acetone – a third ketone body – arises from nonenzymatic
decarboxylation of acetoacetate (Figure 14.11). This decarboxylation reveals the inherent
instability of β-ketoacids. When ketone bodies are elevated, it is possible to detect acetone on
the breath, as acetone is volatile and enters the gas phase (Box 14.3).
Oxidation of ketone bodies (Figure 14.12) occurs in the mitochondria of non-hepatic cells.
First, β–hydroxybutyrate is oxidized to acetoacetate, a reaction catalyzed by β–hydroxybu-
tyrate dehydrogenase, an isozyme of the liver form. The enzyme catalyzing the next step
is absent from the liver, which explains why liver cells cannot oxidize ketone bodies. This
enzyme is succinyl–CoA transferase, catalyzing the reaction of succinyl-CoA with ace-
toacetate, forming succinate and acetoacetyl-CoA. The reaction bypasses the succinyl-CoA
synthetase step of the Krebs cycle, analogous to bypassing P-glycerate kinase of glycolysis
in red blood cells to produce 2,3-bisphosphoglycerate, the regulator of hemoglobin. In both
cases, the bypass incurs an energy cost: ATP in glycolysis and GTP in the Krebs cycle. In
glycolysis, 2,3–bisphosphoglycerate is a regulatory molecule, and the energy cost is kept low
FIGURE 14.10 Ketogenesis I: formation of acetoacetate. These steps of ketone body synthesis
involve joining three acetyl-CoA molecules, forming a four-carbon product – acetoacetate – and
regenerating one acetyl-CoA.
because the flux through the bypass shunt is low. Ketone body oxidation can be a major
pathway, but it leads to the generation of substantial amounts of energy, so its energy cost is
negligible. Acetoacetyl–CoA is subsequently converted to two acetyl-CoA molecules, which
are then oxidized by the Krebs cycle.
Many tissues can utilize ketone bodies for energy; the heart is particularly active. In severe
starvation, the brain can derive a considerable amount of energy by oxidating these com-
pounds, utilizing more ketone bodies than glucose.
14.4 FATTY ACID BIOSYNTHESIS
So far, we have considered acetyl-CoA as the entry point for the Krebs cycle, and the com-
mon intersection of fatty acid and glucose oxidation. In the fed state, when energy storage
336 14.4 Fatty Acid Biosynthesis
FIGURE 14.11 Ketogenesis II: formation of β-hydroxybutyrate and acetone from acetoacetate.
Box 14.3 Acetone Breath
Acetone is the only volatile ketone body, so it is the only one that can be detected by
smelling the breath. Under most conditions, no acetone appears to be present, but in
extreme carbohydrate deficiency combined with an abundance of fatty acids, acetoac-
etate accumulates and acetone is formed nonenzymatically. Acetone imparts a distinc-
tive sweet odor to the breath. Acetone breath is a sign of severe ketoacidosis, the result
of acute alcoholic toxicity, extreme starvation, diabetes mellitus, or diets very low in
carbohydrates.
rather than energy utilization is the physiological direction, acetyl CoA can instead be con-
verted into fatty acids. As all acetyl-CoA formation occurs in mitochondria, but the synthesis
of lipids takes place in the cytosol, there must be a means of exporting carbons for fatty acid
synthesis.
We can conceptually divide the pathway of fatty acids synthesis into three stages
(Figure 14.13):
(a) Export of acetyl-CoA to the cytosol.

(b) Carboxylation of acetyl-CoA to malonyl-CoA.
(c) Sequential addition of two-carbon fragments to form palmitate.
14.4.1 EXPORT OF ACETYL-COA TO THE CYTOSOL
The export of acetyl-CoA to the cytosol is achieved indirectly via the citrate shuttle
(Figure 14.14). Essentially, acetyl-CoA produced in the mitochondria is carried by citrate into
the cytosol. Mitochondrial citrate is formed from the condensation of acetyl-CoA with oxa-
loacetate in the citrate synthase reaction, encountered previously as part of the Krebs cycle.
A selective transporter in the mitochondrial inner membrane enables export of citrate to
the cytosol. Cytosolic citrate is a substrate for citrate lyase, and the products are acetyl-CoA
and oxaloacetate. This reaction involves ATP cleavage and is distinct from the mitochon-
drial citrate synthase reaction. Phosphorylation of the 1-carboxyl group of citrate yields the
FIGURE 14.12 Ketone body oxidation.
acid phosphate (Figure 14.15a). The acid phosphate is then displaced by CoA-SH, leading to
citryl-CoA (also an intermediate of citrate synthase). The steps leading to acetyl-CoA and
oxaloacetate (Figure 14.15b) are familiar ones: acid–base catalysis and C-C bond cleavage.
While acetyl-CoA is destined for fatty acid synthesis, oxaloacetate – the other citrate
lyase product – continues through the citrate shuttle (Figure 14.14). First, a cytosolic isozyme
of malate dehydrogenase catalyzes the conversation of oxaloacetate to malate, using NADH.
Next, malate is oxidatively decarboxylated to pyruvate, catalyzed by malic enzyme. The
electrons from malate are captured as NADPH, the reducing agent used in the latter steps of
fatty acid synthesis. The malic enzyme mechanism is the same as that of the other two oxida-
tive decarboxylation reactions: isocitrate dehydrogenase (in the Krebs cycle) and 6-phospho-
gluconate dehydrogenase (in the pentose phosphate shunt). The three intermediates formed
FIGURE 14.13 General steps in fatty acid biosynthesis. (A) Export of mitochondrial acetyl-CoA
to the cytosol, carried by citrate. (B) Carboxylation of acetyl-CoA. (C) The joining of one acetyl-
CoA with the sequential input of seven malonyl-CoA molecules, and the release of seven CO2
molecules forms palmitate.
FIGURE 14.14 Citrate export shuttle. Acetyl-CoA reacts with oxaloacetate (OAA) to form citrate in the mitochondria. This
citrate is then transported into the cytosol, where it regenerates acetyl-CoA via the citrate lyase reaction. The oxaloacetate prod-
uct is reduced by NADH to malate, which is then decarboxylated at the malic enzyme reaction to pyruvate. The entry of pyruvate
into the mitochondria and carboxylation to oxaloacetate completes the cycle. The net reaction is the movement of mitochon-
drial acetyl-CoA to the cytosol and the conversion of NADH to NADPH through the two cytosolic redox reactions.
FIGURE 14.15 Citrate lyase mechanism. (a) Citryl-CoA formation involves an acid phosphate
intermediate. (b) The splitting portion is initiated by proton abstraction and electron migration,
producing the intermediate carbanion, which becomes protonated to acetyl-CoA.
after oxidation of the alcohol and the electron rearrangements leading to decarboxylation are
compared in Figure 14.16.
Once pyruvate has been formed, it can re-enter the mitochondria and reform oxaloacetate
to complete the cycle. Just as we have seen in the Krebs cycle, the citrate shuttle accepts
acetyl-CoA as a two-carbon input. Whereas the Krebs cycle produces two net CO2 molecules
per turn, the citrate shuttle outputs two carbons per turn in the form of one cytosolic acetyl-
CoA molecule.
14.4.2 CARBOXYLATION OF ACETYL-COA TO MALONYL-COA
The carboxylation of acetyl-CoA, catalyzed by acetyl-CoA carboxylase, is the principal regu-

latory site of fatty acid biosynthesis. Malonyl-CoA formation is the “committed step” in the
formation of fatty acids. The reaction itself is another example of a biotin-dependent carbox-
ylation, with strong similarities to the pyruvate carboxylase reaction. Just as with pyruvate
carboxylase, ATP is used to activate bicarbonate to a carboxyphosphate that can be added to
the biotin cofactor. Bicarbonate from biotin is then donated to the methyl carbon of acetyl-
CoA to form malonyl-CoA at a separate site on the enzyme.
Acetyl-CoA carboxylase (Figure 14.17) is controlled by both allosteric regulation and
covalent modification (protein phosphorylation). The positive regulator, citrate, is a feed-
forward activator of acetyl-CoA carboxylase (similar to the activator of pyruvate kinase,
fructose–1,6–P2). The negative modulator, fatty acyl-CoA, decreases acetyl-CoA carboxylase
activity, reflecting an increase in fatty acids and hence less need for de novo biosynthesis.
Acetyl-CoA carboxylase is subjected to phosphorylation in two ways. One involves cyclic
AMP-dependent protein kinase (PKA) and leads to an inactivation of the enzyme. Since
FIGURE 14.16 Common intermediates of ketoacid decarboxylation. The movement of elec-

trons to release CO2 and subsequently to split a C–B bond is a theme common to isocitrate DH,
6-P-gluconate DH, and malic enzyme.
FIGURE 14.17 Acetyl-CoA carboxylase. The reaction is a carboxylation of acetyl-CoA through

a similar mechanism to pyruvate carboxylase, an intermediate carboxylation of a biotinylated
enzyme. Both allosteric modulation (citrate, fatty acyl-CoA) and phosphorylation (AMPK, PKA)
control the rate of this enzyme.
cAMP is elevated by the starvation-elevated hormone glucagon, PKA-dependent phos-

phorylation and attenuation of liver acetyl-CoA carboxylase is an appropriate response.
Phosphorylation of the liver enzyme is opposed by insulin, which leads to a more active ace-
tyl-CoA carboxylase. The other mode of phosphorylation is through the AMP-activated pro-
tein kinase (AMPK). This enzyme is controlled by changes in AMP levels in cells (Chapter 12)
and causes phosphorylation and inhibition of acetyl-CoA carboxylase. AMPK is a ubiquitous
presence in cells and an important regulator in the liver, adipocyte, brain, and muscle. The
regulatory circuit involving acetyl-CoA carboxylase in lipid metabolism is summarized in
Box 14.4.
14.4.3 SEQUENTIAL ADDITION OF TWO-CARBON FRAGMENTS TO FORM PALMITATE
In mammals, the ensuing reactions of fatty acid biosynthesis are catalyzed by the enzyme
palmitate synthase. Like the pyruvate dehydrogenase complex of mitochondria, the indi-
vidual reaction products remain attached to the enzyme complex. They are passed from one
active site to the next until the final product, palmitate, is released. Structurally, palmitate
synthase in eukaryotes is a dimer in which each strand contains all seven active sites for
the reactions of fatty acid synthesis. These reactions are catalyzed by physically separate,
Box 14.4 Regulatory Circuit of Lipid Metabolism
Several regulatory circuits converge on the enzyme acetyl-CoA carboxylase (CBX), which
in the liver plays a central role in regulating the pathways of fatty acid synthesis, fatty
acid oxidation, and triacylglycerol synthesis (Figure B14.4). Malonyl-CoA, the product of
CBX, inhibits CAT I, the key enzyme required for entry of long-chain fatty acids into the
mitochondria for β-oxidation. Fatty acyl-CoA, the substrate of CAT I, is itself an allosteric
inhibitor of CBX. Other means of regulation of CBX are: allosteric stimulation by citrate,
hormonal regulation (activation by insulin; inhibition by glucagon or epinephrine in adi-
pocytes), and inhibition by 5′-adenosine monophosphate kinase (AMPK). The summation
of cellular events can thus fine-tune the partition of carbon among the lipid pathways.
FIGURE B14.4 Regulatory circuits in lipid metabolism. Pathways are shown in boxes,
enzymes in italics, and intermediates shaded. Allosteric regulation is indicated by dashed lines.
individual enzymes in bacteria. We will examine the reactions under descriptive headers:
loading, condensation, reduction, dehydration, and reduction.
14.4.3.1 LOADING
In preparation for the synthesis, the CoA-SH esters of both malonyl-S-CoA and acetyl-S-
CoA are transferred to the thiol side chain of acyl carrier protein (ACP-SH). The malo-
nyl-ACP product is used for subsequent condensations, as detailed in the following step.
As illustrated in Figure 14.18a, acetyl-S-ACP reacts with a thiol group (cysteine residue) of
acetyl-S-synthase (E-S in the figure), releasing ACP-SH. This reaction leaves the two-carbon
acetyl moiety attached to the enzyme complex, which becomes the first two carbons incor-
porated into the fatty acid. They comprise terminal carbons at the methyl end of the finished
fatty acid.
14.4.3.2 CONDENSATION
In the condensation step, a two-carbon fragment from malonyl-S-ACP is incorporated into

the fatty acyl chain. As illustrated in Figure 14.18b, decarboxylation of the malonyl-S-ACP
drives electron movement to the carbonyl carbon of the acetyl-S-synthase. The CO2 released
FIGURE 14.18 Fatty acid synthase steps. (a) Loading reactions convert the CoA thioesters of
acetyl-CoA and malonyl-CoA to thiol esters of acyl carrier protein (ACP). The acetyl-ACP then
attaches to an -SH group of the synthase (E). (b) Condensation reactions combine an acetyl group
and malonyl-ACP; the latter releases CO2. (c) The first reduction step is the NADPH-dependent
conversion of the carbonyl to an alcohol. (d) In the dehydration step, water is removed to form
the enoyl-CoA. (e) In the second reduction step, the double bond is saturated using electrons
from NADPH.
is the same carbon that was added in the acetyl-CoA carboxylation reaction. Thus, no net
CO2 is incorporated in this pathway, as expected in animal cells. CO2 release provides a ther-
modynamic drive for the reaction (CO2 is a gas and, thus in a separate phase). The condensa-
tion product is β-keto-butyryl-ACP.
14.4.3.3 REDUCTION
The reduction of the carbonyl (Figure 14.18c) is our first encounter with a reaction utilizing
NADPH reducing power. The standard chemical potential of the NADPH/NADP+ couple
is identical to NADH/NAD+, but in cells, the NADPH/NAD+ ratio is more reduced by five
orders of magnitude. This distinction allows NADPH to be used as a reductant for fatty acid
synthesis in the same water space (cytosol) as the more oxidized NADH/NAD+ redox pair
used in reactions related to cellular energy but not biosynthesis.
14.4.3.4 DEHYDRATION
The next step is the removal of the elements of water from the molecule to form a double
bond (Figure 14.18d). This proceeds by the usual route of protonation of the hydroxyl group,
and loss of water coincident with electron migration to form the double bond. The double
bond produced is in the trans configuration, similar to the fumarase reaction of the Krebs
cycle (Chapter 10).
14.4.3.5 SECOND REDUCTION
The double bond is reduced by hydride addition from the cofactor NADPH (Figure 14.18e).
Thus, NADPH is used twice in each round to convert the carbonyl group to a methylene
group. The product is butyryl-S-ACP.
The sequence is repeated in the next round but with a slight variation: the four-carbon
butyryl-S-ACP is used in place of acetyl-S-ACP of (Figure 14.19). In subsequent cycles,
the increasingly larger acyl-ACP is used. Thus, the key steps of fatty acid biosynthesis are
yet another example of repetition–variation, in a similar way to fatty acid oxidation just
described. Once palmitoyl-CoA is formed, a thioesterase specific for the 16-carbon chain
catalyzes palmitoyl-S-ACP hydrolysis to form palmitate and ACP-SH (Figure 14.20).
Cells synthesize various fatty acids starting from palmitate, using distinct enzymatic
reactions, such as those for elongation and desaturation. These enzymes are embedded in
the endoplasmic reticulum. They use acetyl-CoA for elongation and flavin-dependent non-
energy linked oxidation to produce double bonds.
14.5 TRIACYLGLYCEROL FORMATION
Most of the lipid in the body is stored as triacylglycerols. As indicated in Figure 14.21, the
backbone for this class of molecules is glycerol-P, formed in the glycerophosphate DH reac-
tion (Box 14.5). The two hydroxyl groups of glycerol-P accept fatty acyl chains through suc-
cessive acylation reactions with fatty acyl-CoA. Triacylglycerols are molecules with varying
fatty acyl chains that come either from the pathway outlined above (de novo) or from the
dietary route outlined at the beginning of this chapter. The acylation reactions preferentially
incorporate unsaturated fatty acids into the 2-position of the glycerol-P, so that the resulting
triacylglycerols are more unsaturated in this position.
With the two fatty acyl groups present in glycerophosphate, the resulting molecule is
called a phosphatidic acid. The enzyme phosphatidic acid phosphohydrolase catalyzes
removal of the phosphate group, and the products of the reaction are inorganic phosphate
and 1,2–diacylglycerol. Finally, fatty acyl transferase catalyzes the last acylation of a fatty
acyl-CoA to complete the triacylglycerol formation.
14.6 PHOSPHOLIPID METABOLISM
The intermediates of triacylglycerol formation – diacylglycerol and phosphatidic acid –also

lead to phospholipid synthesis. As shown in Figure 14.22, there are two separate routes. The
neutral phospholipid pathway (e.g., phosphatidylcholine formation) has the intermediate
diacylglycerol (DAG), whereas the acidic phospholipid pathway (e.g., phosphatidylinositol
formation) has phosphatidic acid as an intermediate.
An example of the neutral phospholipid pathway is phosphatidylcholine formation, one of
the most common phospholipids in membranes. This is formed by the transferase-catalyzed
reaction of cytidine 5′-diphosphate-choline (CDP-choline) with diacylglycerol (DAG). Notice
that the pathway for triglyceride formation also proceeds from DAG; thus, TG and PC syn-
thesis intersect.
Phosphatidylinositol, an example of the acidic phospholipid pathway, is formed in two
steps from phosphatidic acid. First, a transferase catalyzes the condensation of cytidine
344 14.6 Phospholipid Metabolism
FIGURE 14.19 Chain elongation in fatty acid synthesis. The steps of fatty acid synthase
are repeated with butyryl-ACP in place of acetyl-ACP for the second input of malonyl-ACP.
Subsequent rounds of synthesis follow the pattern indicated here, until palmitoyl-ACP is formed.
FIGURE 14.20 Thioesterase.

FIGURE 14.21 Triglyceride synthesis. After forming glycerol phosphate – the backbone of tria-
cylglycerols by NADH-dependent reduction of dihydroxyacetone-P – two fatty acyl-CoA mol-
ecules are subsequently acylated to the hydroxyl groups of glycerol, forming phosphatidic acid.
The latter is hydrolyzed to diacylglycerol, and a third fatty acyl-CoA is acylated to the backbone.
triphosphate (CTP) with phosphatidic acid to form cytidine diphosphate-diacylglycerol

(CDP-DAG). Subsequently, inositol reacts with CDP-DAG in a synthase-catalyzed reaction to
produce the product, phosphatidylinositol. Both pathways produce cytidine monophosphate
(CMP), but in neutral phospholipid synthesis, the head group (choline in this chase) first
forms a CDP adduct, whereas, in acidic phospholipid synthesis, the phospholipid itself forms
a CDP adduct. Unlike triacylglycerol synthesis, which is limited mostly to liver and adipose
cells, virtually all cells engage in membrane turnover; thus, most cells have significant phos-
pholipid synthesis activity.
346 14.7 Cholesterol Metabolism
Box 14.5 Origins of the Triacylglycerol Backbone
Glucose is an established source of carbon for the glycerol backbone of triacylglycerols

in mammals (Figure B14.5). However, an alternative route is glyceroneogenesis, the ini-
tial portion of the gluconeogenic pathway from pyruvate to dihydroxyacetone-P. This
pathway requires the enzyme PEPCK, which is the key liver pathway for gluconeogen-
esis. PEPCK is found in adipose tissue, which, like the liver, has the complete pathway
for triacylglycerol synthesis. Note that adipose cells do not carry out gluconeogenesis
themselves because they do not have the enzymes fructose bisphosphatase or glucose
phosphatase. The reason for the existence of glyceroneogenesis is not known. It may
provide the metabolic flexibility to complete pathways within the fat cell that lead to
Krebs cycle intermediates. As we’ve seen elsewhere, only acetyl-CoA can be oxidized by
the cycle.
FIGURE B14.5 Origins of the glycerol backbone of triacylglycerol. Glycerol-P (GOLP) is the
backbone of triacylglycerols. It can be derived from glucose by glycolysis. GOLP can also be
formed by a portion of the gluconeogenic pathway from pyruvate, involving the enzyme
P-enolpyruvate carboxykinase (PEPCK).
14.7 CHOLESTEROL METABOLISM
Cholesterol is an essential component of plasma membranes and a precursor to the steroid

hormones and bile salts. Intermediates of the cholesterol biosynthesis pathway are precur-
sors to other cellular products, including ubiquinone and lipid modifiers of proteins. The
pathway for cholesterol synthesis, like fatty acid biosynthesis, requires cytosolic acetyl-CoA,
exported from the mitochondria by the citrate export cycle just described. Three acetyl-
CoA molecules join to form HMG-CoA, using the same reactions as those for ketone body
synthesis (Figure 14.10), except that the cholesterol biosynthesis enzymes are cytosolic iso-
zymes. The next step, the reaction catalyzed by the enzyme HMG-CoA reductase, is rate
limiting for this pathway.
As the control point of cholesterol synthesis, HMG-CoA reductase is a reasonable drug
target for the treatment of hypercholesterolemia. The first clinically effective inhibitor of this
enzyme was lovastatin, a natural product isolated from a fungus. As shown in Figure 14.23
the drug is a precursor to the acid form that bears a strong structural resemblance to HMG-
CoA, as expected of a competitive inhibitor. This drug, and similar ones that have been dis-
covered subsequently, are collectively known as statins.
Figure 14.24 shows a continuation of the cholesterol pathway up to the formation of the
five-carbon intermediate isopentenyl pyrophosphate (isopentenyl-PP). After a reduction of
HMG-CoA to mevalonate, a series of three phosphorylation reactions ensue. The last also
involves a decarboxylation. Its mechanism should be familiar because it has an acid phos-
phate intermediate (like asparagine synthetase) and it removes water (like enolase).
FIGURE 14.22 Phospholipid biosynthesis. There are two separate routes of phospholipid synthesis. Neutral phospholipids like
phosphatidyl choline use preformed CDP-choline, which reacts with diacylglycerol (DAG) to form the product. The triglyceride
synthesis pathway also leads to DAG formation. Acidic phospholipids like phosphatidylinositol involve CDP-DAG formation, which
is displaced by the phospholipid head group inositol to form the product.
FIGURE 14.23 Lovastatin as an HMG-CoA analog. Lovastatin is a lactone. Once inside cells,
esterases catalyze the conversion (hydrolysis) of the lactone to the free acid, which is chemically
similar to HMG-CoA.
FIGURE 14.24 Formation of isopentenyl-PP from HMG-CoA. The first step, catalyzed by HMG-CoA reductase, uses two NADPH
molecules and forms mevalonate, rate-limiting for cholesterol synthesis. Subsequent steps are phosphorylations and a decarbox-
ylation, leading to isopentenyl-PP, the building block for cholesterol as well as other branched lipids such as ubiquinone.
Figure 14.25 outlines the steps from isopentenyl-PP to cholesterol. First, an isomerase
catalyzes the formation of the dimethylallyl-PP. The mechanism is distinct from the isom-
erase reactions we have encountered previously, such as triose-P isomerase (Chapter 9). The
branched lipid isomerase has a carbocation intermediate (Figure 14.26). Enzymatic proton-
ation of the terminal methylene carbon of isopentenyl–PP leads to the intermediate carboca-
tion. The second step, proton abstraction and electron rearrangement in the direction of the
carbocation, leads to the product dimethylallyl-PP. For comparison, the essential mechanism
of the triose-P isomerase is included as a boxed inset in Figure 14.26. The isomerases have
acid–base catalysis in common, but only the branched chain isomerase has a tertiary carbon
that permits stabilization of the intermediary carbocation.
The mechanisms of the ensuing condensation reactions of Figure 14.25 also proceed
through carbocation intermediates. The pyrophosphate end of one precursor is called the
head, and the methylene end is ca1led the tail so that these joining reactions are either head-
to-tail or head-to-head. The formation of geranyl pyrophosphate is a head-to-tail reaction.
Similarly, geranyl-PP reacts with isopentenyl-PP in another head-to-tail reaction to form the
15-carbon farnesyl-PP. Two farnesyl groups condense in a head-to-head reaction to produce
the 30-carbon squalene. In this case, both pyrophosphate groups are released.
The pathway omitted between squalene and cholesterol is complicated. The first two steps
involve a cyclization in which squalene reacts with water to form a multicyclic epoxide and
then an alcohol, a process suggested by the concerted electron flow illustrated in Figure 14.27.
The four-ring structure of lanosterol is also found in cholesterol itself, although several redox
and isomerase steps intervene. In fact, there are two separate pathways established for this
conversion. Recent evidence suggests that the specific route of conversion from lanosterol to
cholesterol is a mixture of the two pathways that varies with cell type.
Cholesterol can be incorporated directly into the plasma membrane. Alternatively, cho-
lesterol can be esterified (often to the fatty acid oleate) and packaged into lipoprotein par-
ticles. The lipoproteins include very low density lipoproteins (VLDL) for export from the liver
and chylomicrons for export from intestinal cells. Cholesterol is also converted into other
products, albeit in lower amounts, such as the bile salt cholate.
COO
H3C
OH
CH3
H
CH3 H
HO OH
H
Cholate
FIGURE 14.25 Isopentenyl-PP to cholesterol. This abbreviated version of cholesterol forma-

tion shows the joining of the five-carbon building blocks to produce the 10-carbon geranyl-PP
and the 15-carbon farnesyl-PP. The latter condenses with itself to form squalene, the last linear
intermediate of cholesterol biosynthesis.
The hydroxylation steps of cholesterol and its derivatives, such as those evident in cholate,
are catalyzed by the enzyme cytochrome P450. This reaction is a mixed-function oxidase,
a family of enzymes that use O2 as substrate. One of the oxygen atoms is incorporated as a
hydroxylation, and the other appears in the product H2O. Several isoforms of this enzyme
exist in liver and are also involved in the hydroxylation of xenobiotics (foreign compounds,
such as drugs). The resulting hydroxylation aids their excretion from the body, both directly
by increasing their solubility and indirectly by providing glycosylation sites.
Bile salts, such as cholate, have all of their hydrophilic groups on one face of the molecule.
Cholate is an amphiphile able to form mixed micelles in lipid digestion described at the
beginning of this chapter. Most of the bile salts secreted into the intestine are reabsorbed
FIGURE 14.26 Mechanism of isopentenyl-PP isomerase. The isomerase catalyzes an acid–

base reaction featuring a carbocation intermediate, unlike the analogous mechanism of triose-P
isomerase of glycolysis (boxed insert).
FIGURE 14.27 Cyclization of squalene to lanosterol. Concerted electron rearrangement and

attachment to the oxygen of water leads to cyclization. An intermediate epoxide forms before
the alcohol lanosterol is produced.
back into the blood, removed by the liver, and exported back through the bile (the hepatobili-
ary circulation). However, a small fraction (<10%) escapes reabsorption and is excreted. This
activity is the only route of cholesterol elimination from the body.
Figure 14.28 presents several products of cholesterol, synthesized by other cells of the
body. For example, Vitamin D3 (dihydroxycholecalciferol) leads to increased formation of a
transport protein that moves Ca2+ into intestinal epithelia and subsequently into the blood-
stream. Estradiol and testosterone are naturally occurring sex hormones. The last entry in
Figure 14.28 is one of the synthetic derivatives of testosterone, an anabolic steroid. First pro-
duced in 1935, these compounds were initially synthesized to mimic testosterone’s ability to
reverse muscle wasting. Various modifications to the molecule enabled oral administration,
which is not possible for testosterone itself. Currently, these anabolic steroids are better
FIGURE 14.28 Derivatives of cholesterol.
known as drugs of abuse in sports. While they promote muscle growth, severe side effects
are now well established, including enhanced tumor growth.
14.8 OTHER LIPIDS
The lipid species we have examined are those present in the largest amounts. Still, many
minor routes of lipid metabolism have products with profound effects on cellular function.
We consider here the eicosanoids, the sphingolipids, and some unusual fatty acids found in
bacteria.
14.8.1 EICOSANOIDS
The eicosanoids are a large class of relatively short-lived molecules (with half-lives in the
seconds) that are derived from the fatty acid arachidonate. Animal cells do not directly store
this fatty acid, nor do they synthesize it, because they do not have desaturase enzymes that
can introduce a double bond beyond the Δ9 position. Precursors of arachidonate, such as lin-
oleic acid, are derived from plants in the diet, so they are called essential fatty acids. Once
arachidonate is synthesized from them by elongation reactions, this fatty acid is incorporated
into plasma membrane phospholipids in the 2-position. Production of arachidonate acid
requires the action of phospholipase A2 (Figure 14.29). Once freed from the phospholipid,
arachidonate has two possible fates, illustrated by examples shown in Figure 14.30. Cyclized
molecules such as the prostaglandin PGE1 require the action of cyclooxygenase, an enzyme
352 14.8 Other Lipids
FIGURE 14.29 Phospholipase A2 reaction
FIGURE 14.30 Arachidonate products. PGE1 is an end-product of the cyclooxygenase path-

way. Leukotriene C4 is an end-product of the lipoxygenase pathway.
irreversibly inhibited by aspirin (acetylsalicylic acid). Blocking this branch of arachidonate

metabolism inhibits the inflammation-inducing prostaglandins.
The non-cyclic branch of arachidonate metabolism leads to the formation of leukotri-
enes, such as the one illustrated in Figure 14.30. These molecules were historically known as
the slow-reacting substances of anaphylaxis, as they stimulate contraction of the airway
ducts in the lung.
There are dozens of eicosanoids, and they affect many physiological pathways, including
muscle contraction, pain, and blood clotting. These molecules act by modulating ongoing
hormonal signaling systems, such as altering the activity state of G-proteins.
14.8.2 SPHINGOLIPIDS
The sphingolipids are a broad class of lipids having serine in place of glycerol as a backbone.
Their presence in the plasma membrane modifies membrane-bound proteins, attaches to the
extracellular matrix, and participates in cell–cell communication. Like the common phos-
pholipids, sphingolipids can be precursors to signal molecules, such as sphingosine-1-P, an
immune cell modulator:
NH2
HO
O
P
O
OH
OH
Sphingosine-1-P
The first step in the sphingolipid pathway is a condensation of serine with palmitoyl-CoA,
catalyzed by serine palmitoyltransferase (Figure 14.31). The intermediate 3-ketosphinganine
is converted after a few steps to ceramide. Ceramide is chemically similar to diacylglyc-
erol; the two often copurify in chromatography. Accordingly, reactions similar to those of
phospholipids produce the major sphingolipids shown in Figure 14.31: sphingomyelin (from
phosphatidylcholine) and the cerebrosides (from UDP sugars such as UDP-galactose). The
sphingolipids have several biological roles, including cell growth, migration, and the regu-
lation of apoptosis. Several pathological states are associated with the inability to degrade
these molecules, such as Neiman–Pick disease, which causes marked mental retardation.
Sphingolipids make up a substantial portion of the lipid sheath (myelin) that wraps nerve
fibers (axons), providing electrical insulation that enables rapid conduction of nerve impulses.
14.8.3 UNUSUAL BACTERIAL FATTY ACIDS
Two of the fatty acids found in bacterial lipids are illustrated in Figure 14.32. The first, an
iso-Cl7:0 is a branched-chain fatty acid that uses carbon from branched-chain amino acids
(e.g., isoleucine, leucine, or valine) as precursors. The other is a cyclopropane-containing
fatty acid, which resembles the conformation of the cis-unsaturated fatty acids such as oleate.
Both modifications alter membrane fluidity in response to the bacteria’s environment. For
example, bacteria synthesize cyclopropane-containing fatty acids when subjected to acidic
media.
14.9 OVERVIEW OF LIPID METABOLISM IN THE FED AND FASTED STATES
In the fed state (Figure 14.33), dietary lipid is packaged into the lipoprotein chylomicron in
the cells lining the intestine (epithelia). Ultimately, the lipid is stored in the adipocyte as a
fat droplet occupying most of the cell volume. The other lipoprotein molecule, VLDL, is
very similar in composition to chylomicrons, with approximately the same density (reflect-
ing the high triglyceride content). VLDL is formed in the liver, as glucose is converted to
fatty acids through fatty acid synthesis, and the fatty acid is packaged with a protein coating
(apolipoprotein). In both humans and murine species, the liver is the prime organ of fatty
acid biosynthesis. Thus, VLDL represents endogenous fat synthesis. Like the triglyceride of
chylomicrons, VLDL triglyceride is destined for the adipocyte triglyceride pool. The major
hormone responsible for shunting lipid metabolism into these pathways is insulin, which
stimulates fatty acid synthesis in the liver and adipocytes. Insulin promotes triglyceride for-
mation from fatty acids in adipocytes.
In the fasted state (Figure 14.34), the major metabolic events involve adipocytes, liver,
and muscle. The adipocyte produces fatty acids through triglyceride breakdown, which is
354 14.9 Overview of Lipid Metabolism in the Fed and Fasted States
FIGURE 14.31 Sphingolipid metabolism. The first reaction, which is rate-limiting for the path-
way, is catalyzed by serine palmitoyl transferase, leading to 3-ketosphinagnine, the backbone for
further acylation. Desaturation and N-acylation leads to ceramide, which can either react with
phosphatidyl choline to form sphingomyelin or with UDP-galactose to form a cerebroside. Due
to the absence of an acyl group at the first position, and the presence of an amide at the second,
these molecules are more stable membrane lipids than the major phospholipids.
stimulated by epinephrine. The liver takes up fatty acids and either completely oxidizes them
to CO2 or partially oxidizes them to ketone bodies. Ketone bodies and fatty acids can be used
by muscle for energy (shown in the figure as conversion to CO2).
A focus on the key role of malonyl-CoA is outlined in Figure 14.35. Here we see multiple
means of regulating the acetyl-CoA carboxylase reaction, primarily important in liver and
muscle. Malonyl-CoA in both muscle and liver is an important regulator of CAT I and there-
fore of fatty acid oxidation. However, malonyl-CoA is a significant precursor to fatty acids
only in the liver. Finally, only the adipocyte provides fatty acids directly for use by other
FIGURE 14.32 Bacterial fatty acids
FIGURE 14.33 Lipid metabolism in the fed state. The intestine provides carbohydrates and
lipid. Carbohydrates can be converted to lipids in the liver, packaged into VLDL, and transported
to the adipocytes for storage as triacylglycerols. Dietary lipids can be packaged into chylomi-
crons and also taken into adipocytes for storage as triacylglycerols.
tissues through control of hormone-stimulated lipase, which is stimulated by epinephrine

and inhibited by insulin. These differences reflect the distinct roles of the cell types: adipo-
cytes store and release fat; muscle utilizes fat and ketone bodies for energy; and the liver is
involved in fat synthesis, VLDL secretion, fatty acid oxidation, and ketone body synthesis.
14.10 INTEGRATION OF LIPID AND CARBOHYDRATE METABOLISM
We have already observed several metabolic connections between lipid and carbohydrate
metabolism. With the major energy-linked pathways now in hand, we will consider three
356 14.10 Integration of Lipid and Carbohydrate Metabolism
FIGURE 14.34 Lipid metabolism in the fasted state. In the fasted state, adipocytes catalyze
the hydrolysis of triacylglycerols to fatty acids, hormonally stimulated by the presence of epi-
nephrine. Blood fatty acids are oxidized in the liver (a process stimulated by glucagon) com-
pletely to CO2 and partially to ketone bodies. Muscle tissue can oxidize fatty acids and ketone
bodies to CO2.
FIGURE 14.35 Control of fatty acid synthesis and oxidation. Malonyl-CoA is formed from ace-
tyl-CoA in the acetyl-CoA carboxylase (CBX) reaction, where it can be further transformed into
fatty acids in the liver and adipose cells. Malonyl-CoA is also an allosteric inhibitor of CAT-I, which
permits long-chain acyl-CoA entry into mitochondria for oxidation. Several factors influence CBX,
including insulin, glucagon, epinephrine, and intracellular AMP concentration. In the adipocyte,
epinephrine stimulates triglyceride hydrolysis, whereas insulin inhibits the process.
situations: the feeding-fasting transition, the resting-exercise transition, and the metabolic
events in diabetes mellitus.
14.10.1 LIPID AND CARBOHYDRATE INTERSECTIONS IN THE FEEDING-FASTING TRANSITION
The fed state is characterized by storage routes for the body’s two principal fuels: glycogen for
sugars and triglyceride for fatty acids. During feeding, elevated insulin concentrations drive
these routes by the uptake of glucose, its conversion to glycogen as well as its conversion to
fatty acids and, thus, triacylglycerols. Due to the ongoing fatty acid synthesis derived from
glucose, malonyl-CoA levels are high, while fatty acid oxidation and ketone body formation
rates are minimal. Fatty acids are shunted into triacylglycerols, and thus their blood levels
are low.
In the fasted state, glucagon concentration becomes elevated, leading to liver glycogen
breakdown to provide blood glucose, and fatty acid synthesis is diminished by inhibiting
acetyl-CoA carboxylase. Malonyl-CoA concentration is depressed, lifting the inhibition of
fatty acid oxidation, increasing ketone body formation. As the epinephrine concentration in
blood is elevated by fasting, fat cells release more fatty acids to the blood, elevating their con-
centration, and in this way, increasing fatty acid oxidation. Maintenance of the blood glucose
concentration is essential to some tissues, including the brain, so gluconeogenesis becomes a
critical pathway. Ultimately, the only substrate for that pathway in starvation is amino acids.
14.10.2 LIPID AND CARBOHYDRATE INTERSECTIONS IN

THE RESTING–EXERCISE TRANSITION
During exercise, blood flow in the body is shunted away from most tissues except for an
increased delivery to muscles and a constant supply to the brain. Thus, we can expect that
most of the metabolic changes involve those of the muscle. In anaerobic exercise, the muscle
breaks down its glycogen store, stimulated by intracellular Ca2+, which also triggers the con-
tractile process. Glycogen is broken down as a result of Ca2+ stimulation of glycogen phos-
phorylase (Chapter 13); during anaerobic exercise, however, lipid metabolism is not affected
by Ca2+. In aerobic exercise, a lipid contribution emerges, as fat cells under the influence of
an increasing epinephrine concentration release fatty acids, which are oxidized by muscle
mitochondria. Reduced malonyl-CoA concentration in muscle is driven by an increased
AMP kinase activity through an increased AMP concentration. Muscle has little fatty acid
synthesis, so acetyl-CoA carboxylase in these cells is exclusively for malonyl-CoA formation
to regulate fatty acid oxidation.
14.10.3 LIPID AND CARBOHYDRATE INTERSECTIONS IN DIABETES MELLITUS
Diabetes mellitus (commonly, just diabetes) is characterized by an elevated blood glucose

concentration and is caused by a relative deficiency in insulin concentration. In type 1 dia-
betes (formerly known as juvenile or insulin-dependent diabetes), the pancreatic beta cells
are impaired, resulting in insufficient insulin release. In type 2 diabetes (popularly known
as adult-onset or non-insulin-dependent diabetes), normally insulin-responsive tissues are
insulin-resistant. Consequently, blood insulin levels can be elevated despite inadequate
amounts of the hormone for action at target cells.
In both cases, the metabolic picture is similar, although, in type 1, the symptoms are
more severe. Due to the relative lack of insulin, the cells respond as they do in starvation:
fatty acids are released from fat cells, ketone body levels are elevated, and gluconeogenesis
is increased. Gluconeogenesis, combined with a depressed glucose uptake into muscle, leads
to the elevated blood glucose concentration that is a signature of diabetes. Insulin therapy is
essential for type 1 diabetes, and is sometimes used for the more commonly occurring type 2
diabetes, although type 2 is more commonly treated by drugs such as metformin. This drug
inhibits liver gluconeogenesis and activates muscle glucose uptake through the activation of
358 Summary
AMP kinase. Exercise leads to the stimulation of AMP kinase as well, which explains why
increased exercise reduces the severity of diabetes, especially for type 2 patients.
While carbohydrates and lipids account for the bulk of the nutrients for mammals, there
are critical roles for amino acids, such as that in nitrogen metabolism, the topic of the next
chapter.
SUMMARY
Animals obtain lipids through the diet (exogenous lipids) or de novo synthesis from carbo-
hydrates and amino acids (endogenous lipids). The dietary route involves forming a mixed
micelle in the intestine with bile salts, cholesterol derivatives synthesized in the liver. Most
of the dietary lipid appears in the blood as chylomicrons, a spherical lipoprotein particle with
a core of triacylglycerols and cholesterol esters and a shell of protein (apolipoproteins) and
phospholipids. Lipids are stored in the adipocytes, and they are released in times of need as
free fatty acids, complexed to the protein albumin in the blood. Most fatty acids are oxidized
to acetyl-CoA in three steps: activation to an acyl-CoA ester, transfer into the mitochondria,
and β–oxidation to produce acetyl-CoA. The Krebs cycle enzymes catalyze the complete
oxidation of acetyl-CoA to CO2. A further metabolic possibility exists for the liver, where
acetyl-CoA can be converted to ketone bodies. Transport into the mitochondria, the middle
step in fatty acid oxidation, is catalyzed by carnitine acyltransferase I (CAT I) and allosteri-
cally inhibited by malonyl-CoA.
Malonyl-CoA, an intermediate of fatty acid biosynthesis, is formed from cytosolic acetyl-
CoA. This acetyl-CoA is derived from mitochondrial citrate exported to the cytosol and
then converted to acetyl-CoA and oxaloacetate. The carboxylation of acetyl-CoA to mal-
onyl-CoA is catalyzed by acetyl-CoA carboxylase, the key regulatory enzyme of fatty acid
biosynthesis. The carboxylase is inhibited by protein kinase A and AMP kinase. Following
this reaction, the complex fatty acid synthase is responsible for seven sequential enzymatic
reactions that lead to palmitate formation. In preparation, acetyl-CoA and malonyl-CoA
esters are converted to thioesters of the protein ACP. Acetyl-ACP reacts with an -SH group
of the fatty acid synthase. Next, malonyl-ACP decarboxylation is linked to its covalent
attachment to the acetyl moiety to produce acetoacetyl-ACP. Reduction (using the cofactor
NADPH) and dehydration steps lead to a butyryl-ACP intermediate. In the next cycle, the
butyryl-ACP adds to the -SH group of fatty acid synthetase. The subsequent steps are reduc-
tion and dehydration; ultimately, a 16-carbon fatty acyl-enzyme intermediate is formed that
is released as palmitate. Ancillary reactions produce other fatty acids of different chain
lengths or with double bonds.
Ketone body synthesis, an exclusive liver pathway, represents a spillover pathway of excess
acetyl-CoA that cannot be oxidized. The ketone bodies, acetoacetate and β–hydroxybutyr-
ate, are utilized for energy by muscle and other tissues.
Triglyceride and phospholipid synthesis pathways involve fatty acyl-CoA addition to the
hydroxyl groups of glycerol-P, producing phosphatidic acid. Hydrolysis of the phosphate of
phosphatidic acid forms diacylglycerol. The subsequent addition of a third fatty acyl-CoA
produces a triacylglycerol. Phospholipids are produced by addition to either phosphatidic
acid (acidic phospholipids) or diacylglycerol (neutral phospholipids) in reactions using CDP-
activated precursors.
The first steps of the cholesterol pathway are the condensation of three acetyl-CoA groups
to form hydroxymethyl-CoA (HMG-CoA), just like the ketone body pathway, except that
cholesterol synthesis takes place in the cytosol. The next reaction of cholesterol synthesis
is catalyzed by HMG-CoA reductase, the rate-limiting step of the process, and the site of
inhibition of cholesterol-lowering drugs such as lovastatin. From this point, cholesterol bio-
synthesis proceeds through the formation of the five-carbon branched intermediate isopen-
tenyl-PP. Several condensation reactions produce the 30-carbon branched lipid intermediate
squalene; after cyclization and several more reactions, cholesterol is formed.
The major pathways of lipid metabolism – fatty acid oxidation, ketone body synthesis, and
fatty acid synthesis – interact with carbohydrate metabolism in normal metabolic processes
(such as fasting and exercise) and disease states (such as diabetes). In general, when energy is
needed for an extended period (e.g., longer than several minutes), lipid metabolism, mainly
through fatty acid oxidation, meets that need. Central to the control mechanisms are the
hormones insulin, glucagon, and epinephrine, and the metabolic intermediate and CAT-I
inhibitor, malonyl-CoA.
REVIEW QUESTIONS
1. Fatty acid synthetase of bacteria consists of seven individual proteins but catalyzes
the same reaction. What would be a plausible explanation for this?
2. Desaturation reactions that produce double bonds following fatty acid synthesis are
not linked to energy formation but still utilize oxygen. Why doesn’t this compete with
mitochondria and potentially impair ATP formation?
3. The mitochondrial oxidation of fatty acids is sometimes called a spiral pathway.
Explain how this is consistent with the description in the text of repetition–variation.
4. A separate class of phospholipids is derived from DHAP directly instead of GOLP and
forms a membrane lipid with an ether-linked fatty acid rather than an ester-linked
one in the 1–position. What would you predict about the chemical stability of these
ether phospholipids?
5. In the presence of fatty acids, lactate carbon is not oxidized for energy to support
gluconeogenesis. Instead, lactate carbon is converted to glucose, called the glucose
sparing effect of fatty acids. How do you think fatty acids can do that?
6. In the fed state, glucose uptake in the liver decreases the oxidation of fatty acids. How
do you think glucose can do that?
7. In sphingolipid metabolism, the production of cerebroside requires the cosubstrate
UDP–galactose. This activated sugar could be provided by galactose metabolism, as
described in the previous chapter. However, suppose galactose is not available in the
diet. What other way could UDP-galactose be produced?
CHAPTER 14 ADDENDUM: THE ISOPRENES
In this chapter, we have considered the isoprene pathway mainly as an intermediate in the
synthesis of cholesterol. A separate metabolic route of the isoprenes was first recognized
in experiments showing that the growth of isolated cells blocked by HMG-CoA inhibitors
(statins) could be relieved by adding back mevalonate but not cholesterol. This finding sug-
gests that intermediates of the branched-chain lipid pathway are essential for cell growth.
The isoprenoids farnesyl-PP and geranylgeranyl-PP can covalently bind certain cellular
proteins, a process known as prenylation. A lipid-protein entity that explains the growth
inhibition is the ras protein, involved in both growth and in tumor formation. Ras is also
the name of a large family of proteins, known as small G proteins. Like the G-proteins
described in Chapter 13, small G-proteins are active when bound to GTP and inactive when
bound to GDP. However, the ras proteins are monomeric and modified by prenylation. The
trimeric G-proteins are also partly prenylated, but its subunits are also modified by palmi-
toylation. In all cases of G-proteins, the attachment of lipid accounts for the ability of these
proteins to act strictly at the surface of the membrane.
The incorporation of farnesyl-PP into a protein is illustrated in Figure CA14.1. Most
proteins covalently modified by prenylation have a pattern of four amino acids at the car-
boxy-terminal, known as a CaaX box. The cysteine (C), is followed by two aliphatic residues
(symbolized by a), and X, representing one of a few other carboxy-terminal amino acids.
The first reaction is catalyzed by farnesyl transferase, attaching the protein to the lipid, and
releasing pyrophosphate. Next, a protease removes the three carboxy-terminal amino acids.
The final reaction is methylation of the carboxyl group, catalyzed by a methyltransferase. The
cofactor for this reaction is SAM, the common precursor to methylation reactions described
in the next chapter.
360 Key Terms
S
PPi Prot-cys-a-a-X
SH
Prot-cys-a-a-X
(SAM)
a,a,X
S
Prot-cys-Me
FIGURE CA14.1 Protein farnesylation. The cys residue of a protein near its carboxyl termi-
nal end displaces the pyrophosphate of farnesyl-PP, forming the intermediate lipid-protein
adduct. Protease cleavage of the three terminal amino acids and methylation using SAM
(S-Adenosylmethionine) completes this lipid modification.
Other inhibitors used as drugs act on the branched-chain lipid pathway. The bisphos-
phonates, used to decrease the breakdown of bone, are essentially pyrophosphate analogs.
These compounds are phosphates separated by a substituted methylene group, for example,
the chlorine-substituted clodronate:
O Cl O
O P C P OH
O Cl O
Chlodronate
The resemblance to pyrophosphate explains the inhibition bisphosphonates exert on prenyl-

ation reactions; a side effect of these drugs is growth inhibition. From another perspective,
the bisphosphonates and statins may be useful as anticancer drugs.
KEY TERMS
acyl carrier protein
anabolic steroids
apolipoprotein
β-oxidation
bile
bisphosphonates
carnitine shuttle
ceramide
chylomicron
chyme
cyclooxygenase
cytochrome P450
emulsification
essential fatty acids
glyceroneogenesis
insulin-resistant
ketogenic
ketone bodies
leukotrienes
phosphatidic acid
prenylation
statins
type I diabetes
type II diabetes
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Nitrogen Metabolism 15
Take the simple glycerol molecule and nitrosylate the hydroxyl groups. The result is
nitroglycerin,
CH2ONO2
CHONO2
CH2ONO2
Nitroglycerin
a compound with the explosive power to disintegrate boulders and ability to alleviate life-threat-
ening blood vessel collapse. The explosion results from the complete oxidation of the molecule
to nitrogen gas (N2). Nitroglycerin in the body releases nitric oxide (NO), which relaxes arterial
smooth muscles. Many nitrogen containing compounds are more stable than oxygen-containing
ones; for example, an amide is more stable than an ester. We can extend the comparison to the
oxygen and nitrogen molecules: O2 is far for reactive than N2. O2 is considered a corrosive gas,
having unpaired electrons that can combine with other elements to form more stable products,
such as H2O. N2 is classified as an inert gas, a triple-bonded structure with no unpaired elec-
trons, and the stable end-product of explosive materials, like nitroglycerin.
Most of the nitrogen in mammalian species resides in the amino acids of proteins.
Nitrogen metabolism revolves around the amino acids and ammonia, which are precursors
to the nucleotides. To place nitrogen metabolism in the context of all organisms, we begin by
considering the global nitrogen cycle.
15.1 THE NITROGEN CYCLE
Like the carbon cycle in which CO2 is incorporated into a myriad of organic compounds and
ultimately released back into the atmosphere. In the nitrogen cycle, atmospheric N2 forms
carbon-bound products in all organisms and is eventually returned to atmospheric N2. N2
is very stable and constitutes about 80% of the atmosphere. It takes the input of enormous
amounts of energy before N2 can be incorporated into forms useful for living systems.
Figure 15.1a summarizes the nitrogen cycle. The critical portion is the conversion of N2
into ammonia (NH3), a process called nitrogen fixation. This reaction can be accomplished
biologically by only a few organisms, such as the Azotobacter soil bacteria. About 40% of
nitrogen fixation is accomplished synthetically using the Haber process, which combines N2
with H2 to form NH3. Bacteria oxidize NH3 to nitrites and nitrates in a series of reactions
known as nitrification. To complete the cycle, bacterial enzymes convert nitrates to N2,
called denitrification.
Many bacteria (and all plants) can also catalyze the reduction of nitrates and nitrites to
NH3 for subsequent use by all organisms, a process known as assimilation. Most of this
chapter is concerned with reactions that begin with NH3 and occur in animals. However,
several energetic principles that we have already developed are evident in examining the
process of bacterial nitrogen fixation, a key part of the nitrogen cycle.
363
364 15.2 Reaction Types in NH3 Assimilation
FIGURE 15.1 Nitrogen cycle. (a) The global nitrogen cycle. NH3 branch is the entry point for
metabolism. (b) Nitrogenase accepts electrons from NADPH, utilizes ATP energy, and forms NH3
from N2.
An expansion of the process of nitrogen fixation is illustrated in Figure 15.1b. Electrons

flow from NADPH through a short electron transport chain to nitrogenase, an enzyme
complex which also requires the energy of 12 high-energy phosphate bonds from ATP.
Nitrogenase passes electrons to a Mo–Fe–S center, which binds N2 and reduces it in sequen-
tial steps to NH3. A nitrogenase complication is its inactivated by O2, which binds the Fe-S
centers and irreversibly inhibits the enzyme. Some bacteria occupy a niche that excludes oxy-
gen, preventing competition for nitrogenase; these organisms are anaerobes. Others keep
oxygen at a level high enough to provide respiration but low enough to preserve nitrogenase.
For example, Rhizobium bacteria grow in the root nodules of legumes, and the pink color of
these nodules is due to leghemoglobin, which binds and sequesters O2.
15.2 REACTION TYPES IN NH3 ASSIMILATION
As with all metabolic pathways, we can better understand nitrogen metabolism by catego-
rizing reactions as metabolically irreversible or near-equilibrium. However, we can further
classify reactions exchanging nitrogen based on their redox changes (Figure 15.2).
15.2.1 REDOX-NEUTRAL
These are not redox reactions because the substrate and product molecules are in the same
oxidation state. An example is the first entry of Figure 15.2, the hydrolysis of an amide to a
carboxylic acid plus ammonia.
15.2.2 REDOX-ACTIVE
Redox-active reactions are true redox exchanges, so these reactions require an exogenous
electron acceptor. For example, the conversion of an amine to a carboxyl group is an oxida-
tion that produces two electrons.
Chapter 15 – Nitrogen Metabolism 365
FIGURE 15.2 Classifying reactions in nitrogen metabolism by redox changes. Redox-neutral

reactions do not transfer electrons; redox-active reactions require an external mobile carrier for
electrons; and redox-balanced reactions have an oxidized and reduced partner in both the sub-
strate and the product, with no net electron transfer within the reaction.
15.2.3 REDOX-BALANCED
In a redox-balanced reaction, one substrate is oxidized while the other is reduced. The overall
change is two compensating redox reactions, with no net redox change. For example, the
transaminases are redox-balanced reactions.
15.3 METABOLICALLY IRREVERSIBLE NITROGEN EXCHANGE REACTIONS
One metabolically irreversible reaction is catalyzed by glutamine synthetase:

(15.1) Glutamate + ATP + NH 3 ® Glutamine + ADP + Pi
This reaction, in which an amide is formed from a carboxylic acid and ammonia, is redox-
neutral. A separate metabolically irreversible, redox-neutral reaction, catalyzed by glutamin-
ase, is the hydrolysis of the amide bond:

(15.2) Glutamine ® Glutamate + NH 3
Combining the reactions in Equations (15.1) and (15.2) would result in net hydrolysis of ATP,
similar to the substrate cycles of oxidative phosphorylation. However, cells usually do not
contain substantial amounts of both of these enzymes in the same metabolic space, so these
reactions constitute separate metabolic routes rather than a potential regulatory cycle. Only
the release of ammonia or the incorporation of ammonia takes place at one time.
Glutamine serves as a carrier of nitrogen between tissues. During amino acid breakdown
in muscle, glutamine synthetase is active, and the muscle releases glutamine to the blood.
Liver takes up this glutamine and converts it to glutamate and NH3 via the glutaminase reac-
tion. The nitrogen is converted to urea by the pathway we consider in Section 15.4.
A more specialized, metabolically irreversible reaction is catalyzed by monoamine oxidase,
responsible for the inactivation of the neurotransmitters, such as serotonin (5-hydroxytryp-
tamine), shown in Figure 15.3. This nitrogen exchange is redox-active: electrons are extracted
from the monoamine to reduce O2 to H2O2. Monoamine oxidase is embedded in the outer
366 15.4 Near-Equilibrium Nitrogen Exchange Reactions
FIGURE 15.3 The monoamine oxidase reaction.
FIGURE 15.4 Glutamate dehydrogenase
mitochondrial membrane so that functionally it reacts with substrates in the cytosol and is
not linked to energy capture. The enzyme contains a bound FAD cofactor, serially extracting
electrons prior to their donation to O2, with protons balancing the reaction. Most cells carry
out one further catabolic step: the oxidation of the aldehyde product to the acid, an end prod-
uct that exits the cells. Serotonin is a neurotransmitter responsible for digestive motility and
mood changes. Monoamine oxidase catalyzes the degradation and thus inactivation of this
neurotransmitter; inhibitors of this enzyme were once widely used as antidepressants.
15.4 NEAR-EQUILIBRIUM NITROGEN EXCHANGE REACTIONS
Glutamate DH, a near-equilibrium enzyme, plays a key role in nitrogen metabolism. A sec-
ond class of near-equilibrium enzymes with essential roles in nitrogen metabolism are the
transaminases.
15.4.1 GLUTAMATE DH
This reaction, found in the mitochondrial matrix, converts the Krebs cycle intermediate α–
ketoglutarate to glutamate:
(15.3) a-Ketoglutarate + NH 3 + NADH Glutamate + NAD+
The two steps involved in the direction of glutamate formation are shown in Figure 15.4.
The first is the formation of the intermediate Schiff base. In the second, NADH reduces the
double bond. One explanation for ammonia toxicity is the depletion of α-ketoglutarate from
the Krebs cycle, a result of the near-equilibrium nature of glutamate DH. Thus, an appre-
ciable rise in ammonia levels could drive glutamate formation, limiting the concentration
of α-ketoglutarate. Interruption of the Krebs cycle is particularly deleterious for brain cells,
which depend heavily on oxidative metabolism.
15.4.2 TRANSAMINASES
Transaminases (aminotransferases) are enzymes that catalyze the interconversion of amino

acids and keto acids. These are part of the malate/aspartate redox shuttle. The mechanism for
aspartate aminotransferase is illustrated in Figure 15.5. Details of electron flow in reactions
involving pyridoxal-P were presented in Chapter 7. The first step of the aminotransferase
FIGURE 15.5 Mechanism of aspartate transaminase. Aspartate binds the cofactor PALP (pyridoxal-P), forming a Schiff base.
After several electron rearrangement steps, OAA (oxaloacetate) – the ketoacid of aspartate – is released, leaving the N atom
bound to PALP. This is the one half of a symmetrical reaction mechanism. In the remainder (not shown α-ketoglutarate forms a
Schiff base with the pyridoxamine form of the cofactor and is released as glutamate.
reaction is Schiff base formation, which leads to an intermediate stabilized by hydrogen

bonding to the dissociated hydroxyl group of pyridoxal phosphate. Next, electrons flow to
the positively charged ring nitrogen (step 2), an electron sink. In step 3, a partial reverse
flow along the same path creates a new Schiff base, except the pyridoxal phosphate carbon
(starred in Figure 15.5) is at the redox level of methylene. Hydrolysis of the Schiff base (step 4)
releases the ketoacid, oxaloacetate. This step completes the half-reaction, leaving an enzyme-
bound pyridoxamine. In a mirror image to the mechanism just outlined, α–ketoglutarate
forms a Schiff base to the pyridoxamine, extracts the nitrogen, and ultimately is released as
glutamate. Note that there is a redox transfer in each half-reaction because the amine is more
reduced (by two electrons) than the ketoacid. In the complete reaction, there is no net redox
transfer (i.e., it is redox-balanced).
Release of the first product prior to binding of the second leaves the enzyme in the pyri-
doxamine form (Figure 15.5). The enzyme is restored to its initial state only after binding
the second substrate. This type of reaction mechanism is known as ping-pong: the first
substrate binds, the first product is released, the second substrate binds, and the second
substrate is released. By contrast, enzymes with multiple substrates that all bind before any
product is released are called sequential.
Because transaminases catalyze exchange reactions, they do not remove or add nitro-
gen from substrates in a net sense. Instead, they funnel nitrogen into glutamate, a common
amino acid that has broader metabolic connections. For example, the removal of nitrogen
from alanine to form pyruvate plus NH3 is the result of the following two reactions:

(15.4) Alanine + a-Ketoglutarate ® Pyruvate + Glutamate

(15.5) Glutamate + NAD+ ® a-Ketoglutarate + NADH + NH 3
368 15.5 The Urea Cycle
An additional example of the combination of transaminase and glutamate DH reactions is

the urea cycle, which we examine next.
15.5 THE UREA CYCLE
In humans, most of the nitrogen excreted is in the form of urea, synthesized from mixtures
of amino acids and ammonia exclusively in the liver. The pathway for urea synthesis does not
occur in isolation but is tied to gluconeogenesis. Moreover, for any mixture of ammonia and
amino acids presented to the liver, half of the urea nitrogen comes from ammonia, and half
comes from aspartate.
A portion of the pathway (Figure 15.6) emphasizes the transport of aspartate and citrul-
line from the mitochondria into the cytosol, and their condensation in the argininosuccinate
synthase reaction. Viewed in this way, the urea synthesis requirement for exactly the same
amount of aspartate and citrulline (the latter representing the ammonia input) is simply a
reflection of the stoichiometry of cytosolic argininosuccinate synthetase. To see how vary-
ing inputs of substrates are balanced, we need to examine what happens in just two limiting
cases: when NH3 is in excess and when aspartate is in excess.
15.5.1 [NH3] EXCEEDS [ASPARTATE]
With NH3 in excess, glutamate DH runs in the direction of glutamate formation (Figure 15.7a).
Glutamate subsequently reacts with oxaloacetate through the aspartate aminotransferase
reaction to produce the extra aspartate needed. Note that glutamate and α-ketoglutarate are
part of a small cycle in this process, and a net input of oxaloacetate occurs. The oxaloacetate
itself may be derived from pyruvate through the pyruvate carboxylase reaction. The ultimate
fate of this carbon is glucose formation.
FIGURE 15.6 Origin of the nitrogen for the urea cycle. The urea cycle requires mitochondrial
export of equal quantities of aspartate and citrulline. The two condense in the cytosol. Urea
obtains one N atom from aspartate and the other from citrulline. As indicated, the N atom of
citrulline comes from NH3.
FIGURE 15.7 Balancing nitrogen flows for the urea cycle. To match exactly the N from NH3
and aspartate, the near-equilibrium reactions of glutamate DH (GLDH) and aspartate aminotrans-
ferase (AAT) run in different directions in (a) and (b), balancing the forms of nitrogen encountered
by the liver.
15.5.2 [ASPARTATE] EXCEEDS [NH3]
With aspartate in excess, glutamate DH runs in the direction of α-ketoglutarate formation

(Figure 15.7b). α-Ketoglutarate subsequently reacts with aspartate through the aspartate
aminotransferase reaction to regenerate glutamate to complete the small cycle just described
for NH3 excess, but running in the opposite direction. The excess oxaloacetate produced
ultimately is converted to glucose.
The near-equilibrium reactions can adjust to prevailing concentrations of substrates and
to alter their reaction direction, thus matching the concentrations of aspartate and citrulline
(representing ammonia) in the cytosol for urea formation. Like the nonoxidative pentose
phosphate shunt reactions, the notion of near-equilibrium reactions provides a clear under-
standing of how reaction flux can be diverted into separate routes depending only on their
prevailing concentrations. With this background, we can now consider the details of the
cycle itself.
15.5.3 STEPS FROM NH3 TO CITRULLINE
The two reactions shown in Figure 15.8 are required to synthesize citrulline from NH3. First,
NH3, bicarbonate, and two ATP molecules react to produce carbamoyl P. This reaction, cata-
lyzed by carbamoyl-P synthetase, resembles the reaction catalyzed by asparagine synthe-
tase in that one ATP is used to form an acid phosphate that is displaced by ammonia. The
sequence involving carbamoyl-P synthetase is illustrated as an inset to Figure 15.8. A second
ATP reacts to form carbamoyl-P, the acid phosphate product. In the next reaction, catalyzed
by ornithine transcarbamoylase, carbamoyl-P is displaced by the amine of ornithine, a non-
protein amino acid, to form citrulline.
15.5.4 CYTOSOLIC STEPS OF THE UREA CYCLE
With the arrival of citrulline and aspartate in the cytosol, both nitrogen atoms for urea syn-
thesis are represented. The condensation step, catalyzed by argininosuccinate synthetase,
joins the molecules at the expense of two high-energy phosphates; the reaction converts
370 15.5 The Urea Cycle
FIGURE 15.8 Urea cycle: from NH3 to citrulline. The formation of carbamoyl-P, the first step, is
detailed in the inset. Next, ornithine transcarbamoylase catalyzes the addition of carbamoyl-P to
ornithine, forming citrulline.
FIGURE 15.9 Cytosolic steps of the urea cycle. The cytosolic portion of the urea cycle includes the condensation reaction
between citrulline and aspartate, the subsequent formation of arginine with release of fumarate, and the formation of urea.
Ornithine is transported into the mitochondria to complete the cycle.
ATP to AMP and PPi (Figure 15.9). The mechanism is outlined in Figure 15.10. The adenosyl-
P portion of ATP forms a phosphate ester with the ureido [NC(=O)N] group of citrulline.
Next, the amine of aspartate displaces AMP, forming the product argininosuccinate.
Returning to the pathway of Figure 15.9, argininosuccinate lyase catalyzes the hydrolysis
of argininosuccinate to arginine and fumarate. Fumarate is converted to malate through a
reaction catalyzed by cytosolic fumarase, and malate is converted to glucose. Malate could
FIGURE 15.10 Mechanism of argininosuccinate synthetase. After the formation of the acid
phosphate with the ureido group of ornithine, the amine of aspartate displaces AMP to form the
condensation product.
FIGURE 15.11 The urea cycle.
enter the mitochondria but cannot be oxidized by the Krebs cycle because it is an interme-
diate rather than a substrate for this pathway. Thus, the fate of fumarate carbon is glucose
formation.
The other product of the argininosuccinate lyase reaction – arginine – is hydrolyzed to
urea and ornithine, a reaction catalyzed by arginase. The ornithine is transported into the
mitochondria, completing the urea cycle.
15.5.5 OVERALL UREA CYCLE
Figure 15.11 shows the complete urea cycle. The mitochondrial reactions collect nitrogen,
half in aspartate and half in citrulline (representing ammonia). In the cytosol, two mole-
cules exit the cycle: fumarate, destined for glucose synthesis, and urea, which is not further
metabolized in humans. After it leaves the liver, urea enters the kidney, and from there, it is
ultimately excreted from the body. While urea is metabolically inert in the kidney, it forms
an osmotic gradient that concentrates the urine, minimizing water loss.
Regulation of the urea cycle is exerted at three levels. First, near-equilibrium reactions
control nitrogen delivery to urea from various mixtures of amino acids and ammonia.
Thus, as more nitrogen-containing molecules are provided to the liver, more urea is formed.
Secondly, there is allosteric control exerted at carbamoyl-P synthetase. This reaction is
activated by the molecule N-acetylglutamate; its levels correlate to states of increased urea
372 15.6 Amino Acid Metabolism: Catabolism
synthesis (reflecting increased dietary protein). Finally, the protein concentration of the urea
cycle enzymes can be increased under conditions in which more urea formation is required.
This genetic control is a long-term activity that can raise the liver’s upper limit of urea forma-
tion. In fasting conditions, for example, protein breakdown is more extensive, and glucagon
stimulates the liver to synthesize increased concentrations of all of the urea cycle enzymes in
concert with the gluconeogenic enzymes.
15.6 AMINO ACID METABOLISM: CATABOLISM
For the 20 common amino acids, each with synthetic and catabolic pathways, there are 40
separate routes. The metabolic complexity is even greater, considering species differences,
nonprotein amino acids, and existence of alternative pathways. Accordingly, we present only
partial amino acid metabolism pathways, emphasizing general principles.
In amino acid metabolism, it is not always clear which steps can be strictly categorized as
catabolic or anabolic. We will consider the removal of nitrogen and the disposal of carbon as
catabolism. Catabolism can be subdivided into two groups: ketogenic and glucogenic. After
nitrogen removal, the carbon skeleton is converted to acetyl-CoA and potentially ketone
bodies (ketogenic) or a precursor of glucose (glucogenic). Most amino acids are at least par-
tially glucogenic; only a few are exclusively ketogenic. We begin by considering branched-
chain amino acid catabolism, which illustrates routes of structurally related amino acids that
are ketogenic, glucogenic, and both.
15.6.1 BRANCHED-CHAIN AMINO ACID BREAKDOWN
The catabolism of leucine, isoleucine, and valine are similar (Figure 15.12). Each is first
converted to the corresponding ketoacid by a transamination reaction and then to a CoA
thioester through the action of a dehydrogenase complex, which is analogous to the pyruvate
dehydrogenase complex. Branched-chain amino acid transaminases are particularly rich in
muscle tissue, and the dehydrogenase complexes (and subsequent steps) are particularly
enriched in the liver. Oxidation steps following the formation of the CoA thioesters are dis-
tinct for each branched-chain amino acid.
Ultimately, leucine forms acetyl-CoA and is thus a ketogenic amino acid. Isoleucine forms
acetyl-CoA and succinyl-CoA and is therefore partly ketogenic and partly glucogenic. Valine
breakdown leads to succinyl-CoA and is thus glucogenic. A dramatic increase in blood con-
centrations of branched-chain amino acids and their ketoacid forms occurs in maple syrup
urine disease resulting from a deficiency in the branched-chain dehydrogenase complex
(Box 15.1).
15.6.2 THREONINE
In mammals, the major pathway for the degradation of threonine is the conversion to 2–keto-
butyrate, catalyzed by threonine dehydratase (Figure 15.13). The ensuing step is catalyzed by
a dehydrogenase complex similar to the branched-chain dehydrogenases just introduced in
the section above (as well as pyruvate dehydrogenase and α-ketoacid dehydrogenase com-
plexes). The product propionyl-CoA is converted to succinyl-CoA by the pathway described
for odd-chain numbered fatty acid catabolism (Chapter 14). As succinyl-CoA is an inter-
mediate of the Krebs cycle, it cannot be oxidized; instead, it is converted to glucose. Thus,
threonine is a glucogenic amino acid.
15.6.3 LYSINE
The initial stages of lysine degradation (Figure 15.14) are analogous to the first cytosolic reac-
tions of the urea cycle. A condensation step and subsequent splitting results in the transfer
FIGURE 15.12 Branched-chain amino acid catabolism. The three branched-chain amino acids
have a similar catabolic pathway: transamination followed by a dehydrogenase complex that
releases CO2, and subsequent breakdown steps to either acetyl-CoA (Leu and Ile) or succinyl-CoA
(Ile and Val).
Box 15.1 Clinical Cases of Defects in Amino Acid Metabolism
Inborn errors (genetic deficiencies) in amino acid metabolism are common and have dra-
matic consequences. For example, a loss of phenylalanine hydroxylase results in tyrosine
insufficiency and an accumulation of phenylpyruvate in the urine, producing a musty
smell. This disease is called phenylketonuria (PKU). PKU was also one of the first enzyme
deficiencies for which prenatal screening was implemented. Much of the damage from
this disorder can be averted by a strict diet that is low in protein and includes supplemen-
tal tyrosine. Besides mental disorders (phenylalanine is a neurotransmitter precursor), a
prominent feature is the near absence of skin pigmentation because tyrosine is also a
precursor to the dark pigment melanin. Another disorder of amino acid metabolism is
maple syrup urine disease, a result of the inability to oxidize branched-chain amino
acids. Once again, the ketoacid products accumulate in the urine (causing a sweet, syrup-
like smell). Like PKU, this disease also causes mental defects (and a possible coma), and is
treated by a strict dietary restriction of the branched-chain amino acids.
of nitrogen between molecules. Both reactions are catalyzed by the bifunctional enzyme sac-
charopine DH. In the first step a reductase – lysine condenses with α-ketoglutarate, forming
saccharopine. In the next step – a dehydrogenase – saccharopine is oxidized and split into
the products, glutamate and α-aminoadipate semialdehyde. A semialdehyde is a molecule
derived from a dicarboxylic acid, where one of the acid groups has become an aldehyde; in
this case adipic acid is the parent dicarboxylic acid.
FIGURE 15.13 Threonine catabolism. The deaminase step (1) is followed by a dehydrogenase
complex (2), similar to the branched-chain dehydrogenase complexes and pyruvate dehydroge-
nase complex. Ultimately, the pathway leads to succinyl–CoA, so threonine is glucogenic.
Subsequent steps convert the semialdehyde intermediate to acetyl-CoA and acetoacetate;

thus, lysine – like leucine – is a purely ketogenic amino acid.
15.6.4 TRYPTOPHAN
The major end products of tryptophan catabolism are alanine and acetoacetate, making this
amino acid both glucogenic and ketogenic. A branch point in the pathway leads to the forma-
tion of NAD+. However, the amount of this redox cofactor synthesized in humans is insuf-
ficient for metabolic needs, so dietary input (as nicotinamide) is still required.
15.6.5 PHENYLALANINE AND TYROSINE DEGRADATION
The initial stages of the breakdown of phenylalanine are illustrated in Figure 15.15. The first
step is an aromatic ring hydroxylation, catalyzed by phenylalanine hydroxylase. In addition
to the mobile cofactors indicated in the diagram, biopterin cofactors also participate in the
reaction; two are illustrated in Figure 15.16. Tetrahydrobiopterin binds the enzyme along
FIGURE 15.14 Lysine catabolism. The first two steps, (1) condensation of lysine and
α-ketoglutarate to saccharopine and (2) formation of the semialdehyde and glutamate, remove
one nitrogen from the amino acid. Further steps produce only acetoacetate and acetyl-CoA;
thus, lysine is exclusively ketogenic.
FIGURE 15.15 Phenylalanine and tyrosine breakdown.
with O2 and phenylalanine, assisted by a nonheme iron at the active site. One oxygen atom
is incorporated into the amino acid substrate to form the product tyrosine; the other is tran-
siently attached to the biopterin. Further enzymatic steps remove the oxygen as water to
form dihydrobiopterin and reduce it to regenerate tetrahydrobiopterin. Transamination of
tyrosine produces p–hydroxyphenylpyruvate, followed by several steps that lead to fumarate
and acetoacetate. Thus, both phenylalanine and tyrosine are ketogenic as well as glucogenic
amino acids.
FIGURE 15.16 Biopterin structures.
FIGURE 15.17 Direct degradation of amino acids to major pathway intermediates. Single-step
pathways degrade glutamate, aspartate, alanine, glutamine, and asparagine.
15.6.6 AMINO ACIDS DIRECTLY CONNECTED TO MAJOR METABOLIC PATHWAYS
Several amino acids are degraded directly through a transaminase reaction to an intermedi-
ate of a major pathway, usually glycolysis or the Krebs cycle. For example, the reaction cata-
lyzed by glutamate aminotransferase can convert glutamate to α-ketoglutarate or aspartate
to oxaloacetate. Alanine is converted in one step to pyruvate. These reactions are diagramed
in Figure 15.17, which also illustrates the deamination reactions of asparagine and glutamine.
The degradation of arginine to ornithine that we examined in the urea cycle cannot be con-
sidered a true degradative pathway because the product ornithine is part of the cycle. Thus,
while this amino acid is connected to a major metabolic pathway, its complete degradation is
FIGURE 15.18 Amino acid pathways converging on glutamate. Arginine and proline converge
at glutamate semialdehyde, which forms glutamate. Histidine degradation, after the transfer of a
carbon to THF, forms glutamate. All three are thus glucogenic.
not. We consider arginine next, along with proline and histidine, two other amino acids that
are ultimately converted to glutamate.
15.6.7 ARGININE, PROLINE, AND HISTIDINE ARE ALL CONVERTED TO GLUTAMATE
The degradation routes of arginine, proline, and histidine are shown in Figure 15.18. Arginine
breakdown (Figure 15.18a) uses the urea cycle enzyme arginase. The ornithine product is
subsequently converted to glutamate semialdehyde through the action of a transaminase
(Figure 15.18b) and to glutamate through a dehydrogenase reaction (Figure 15.18c).
Proline catabolism converges with the arginine route at glutamate semialdehyde after two
reactions: oxidation to pyrrolidine-5-carboxylate (Figure 15.18d) and hydrolysis of the ring
(Figure 15.18e).
Histidine also is converted to glutamate via the abbreviated route shown in Figure 15.18f.
The proximal intermediate to glutamate is N-formimino-glutamate. The formimino por-
tion is transferred to a cofactor abbreviated THF in the figure, thus forming glutamate.
Tetrahydrofolate (THF) is one of the key cofactors in what is called one-carbon metabolism,
which is considered next in the context of the degradation of other amino acids that are more
closely connected to this process.
15.6.8 ONE-CARBON (1C) METABOLISM AND SERINE,

GLYCINE, AND METHIONINE BREAKDOWN
The cofactor tetrahydrofolate just introduced in histidine catabolism is derived from folic
acid (Figure 15.19a; Box 15.2). Folic acid is a vitamin because it is not synthesized in humans.
Once ingested into the body, folic acid is converted to tetrahydrofolate (Figure 15.19b), a
reduced form of the pteridine, and a polyglutamate tail with two to four additional gluta-
mate residues linked with amide bonds. The N5 and N10 positions (blue in the diagram) are
the one-carbon attachment sites of the molecule (with the “active portion” of the molecule
shown in Figure 15.19c). Either or both of these nitrogen atoms may be bound to a single
carbon unit (or, in some cases, to a formimino group). In amino acid metabolism, the car-
rier molecule (i.e., THF) does not change its redox state; this happens in only one reaction of
nucleotide metabolism (Section 15.8.9, A Unique Methylation to Form dTMP).
The catabolism of serine is illustrated in Figure 15.20. The first reaction, catalyzed by
serine hydroxymethyltransferase, has the prosthetic group pyridoxal phosphate, which first
binds the amine of serine as shown in the diagram. Note the similarity to the intermediate
and subsequent electron flow in the transaminase reactions (Figure 15.5). In the case of the
FIGURE 15.19 Folic acid and THF. (a) Folic acid is the vitamin and precursor to the cofactor, tet-
rahydrofolate (THF). (b) The cofactor THF. (c) The active nitrogen atoms involved in one-carbon
transfer are highlighted.
Box 15.2 Word Origins: Folate
The isolation of a compound from spinach leaves in 1941 was given the name folic acid,
following a long tradition of assigning a name to a molecule based on its biological ori-
gin (e.g., insulin for the “islands” of islet cells; formic acid from the ant [Latin: formica]).
Folic acid is indeed found in leafy vegetables, such as spinach and collard greens, but
also other plants such as lentils. The use of folio is much older, a Latin word referring to
a leaf of paper. Reference to leaves as foliage is still in everyday use. However, signifi-
cant concentrations of folic acid are restricted to a small number of mostly dark-leaved
vegetables.
FIGURE 15.20 Serine and glycine degradation. Enzymes of serine and glycine breakdown
transfer carbons to THF. The intermediate of the transferase reaction is a Schiff base bound to
pyridoxal phosphate, involving electron rearrangement similar to that of aminotransferases.
present enzyme, electron flow leads to splitting of a C–C bond as indicated, and the single
carbon released becomes attached to THF. After rearrangement and hydrolysis, pyridoxal
phosphate is regenerated, and glycine is released. The second reaction (Figure 15.20), cata-
lyzed by the glycine cleavage system, has a mechanism similar to the pyruvate dehydroge-
nase complex and the branched-chain amino acid dehydrogenase complexes introduced in
this section. As sketched in Figure 15.21, the enzyme complex has the same swinging arm,
a lipoamide connected to a central protein (called H), which first reacts with the substrate
glycine at the active site of the P protein (pyridoxal phosphate containing). The glycine-PALP
complex is similar to that of the serine hydroxymethyl-transferase reaction. In this case, CO2
is released, and the resulting fragment becomes attached to the swinging arm as a sulfhydryl
adduct. In the next step (T), the carbon fragment is transferred to THF, leaving the lipoamide
in the reduced form. In the last reaction (L), reduced lipoamide formation is catalyzed by
lipoamide dehydrogenase, the same FAD-bound enzyme found in the other dehydrogenase
complexes. In all cases, electrons are passed to the mobile cofactor NAD+.
380 15.7 Amino Acids: Anabolism
FIGURE 15.21 Glycine cleavage system. The swinging arm mechanism with three separate
enzyme activities in the complex resembles the dehydrogenase complexes for pyruvate and the
branched-chain amino acids.
While there are other routes for serine oxidation, the one described here is the major
pathway in mammals. Thus, in mammals, serine and glycine metabolism are strongly linked.
Methionine degradation is also connected to 1C metabolism. In an unusual addition reac-
tion, methionine reacts with the adenosine moiety of ATP, forming an adduct called S–
adenosylmethionine (SAM; Figure 15.22). The other products of this reaction, catalyzed by
methionine adenosyltransferase, are PPi and Pi. SAM is a methyl donor for a variety of reac-
tions, as indicated in step (2) in which variable acceptors are indicated as an X. After methyl
donation, S-adenosylhomocysteine (SAH) is formed. Some methyl acceptors and products
are shown in Figure 15.23. Methylated products include the hormone epinephrine, the phos-
pholipid phosphatidylcholine, and proteins such as histone, which are commonly methylated
on lysine residues as shown. SAM is also a methyl donor for the nucleic acids.
Reactions 1 to 4 of Figure 15.22 constitute the methionine cycle. This route connects
the 1C metabolism of THF with the methyl donor SAM. The ultimate source of the carbon
may arise from the “loaded” THF pool, much of which can arise from serine and glycine
metabolism, as just described. Various forms of THF can be interconverted. Reaction 4 of
Figure 15.22, catalyzed by methionine synthase, has an additional bound cofactor cobala-
min, or vitamin B12. This prosthetic group has a cobalt ion attached to a ring system much
like the iron ion in the heme group of hemoglobin and the cytochromes. The Co+ of cobala-
min directly binds the carbon involved in 1C transfers. Cobalamin also transiently binds a
methyl carbon in the oxidation of odd-numbered fatty acid chains (Chapter 14). Deficiencies
in folate or vitamin B12 due to dietary insufficiency or inadequate absorption (such as in alco-
holics) cause severe anemias (Box 15.3).
15.7 AMINO ACIDS: ANABOLISM
Biosynthetic pathways of amino acids may be categorized as essential or nonessential

(Table 15.1). The nonessential amino acids are those for which pathways exist in humans,
whereas the essential amino acids must be obtained from the diet. As omnivores, humans
usually have no difficulty getting sufficient amino acids, mostly for protein synthesis and
for other purposes, as described in the following sections. However, vegetarians must con-
sciously combine foods (e.g., rice and beans) to obtain essential amino acids because plant
proteins do not contain the entire complement of amino acids.
FIGURE 15.22 Methionine catabolism and the methionine cycle. Abbreviations: SAM,
S-adenosylmethionine; SAH, S-adenosylhomocysteine. The enzymes are 1) methionine adenos-
yltransferase, 2) methyltransferase, 3) SAHase, and 4) methionine synthase.
FIGURE 15.23 Methylation targets of SAM. The methyl groups in the product are highlighted
in blue.
382 15.7 Amino Acids: Anabolism
TABLE 15.1 Essential and Box 15.3 Vitamin B12 and Folate Deficiencies
Nonessential Amino Acids
The close relationship between THF and cobalamin (Vitamin B12) is evident in the methio-
Essential Nonessential
nine cycle, but their association was noted many years before this pathway was estab-
Cys Ala
lished. Deficiency of either leads to anemias, a common condition in older patients.
His Arg
Moreover, any condition that alters absorption can also lead to a relative folate defi-
Iso Asp
ciency. B12 absorption requires the presence of a protein synthesized in the stomach
Leu Glu
called intrinsic factor (involved in intestinal B12 transport), and a lack of this protein leads
Lys Asn
to a relative deficiency even with normal dietary B12. Cobalamin is not present in plants,
Met Gln
so strict vegetarians run the risk of anemia. Extreme – but increasingly more common –
Phe Gly
measures to resolve severe obesity include surgery, which may involve removing part of
Thr Pro
the stomach and/or intestine, indirectly cause a deficiency in these vitamins. Finally, the
Trp Ser
intake of alcohol, and some drugs, such as metformin (diabetes), proton-pump inhibitors
Tyr –
(acid reflux), and chloramphenicol (an antibiotic) interfere with the uptake of B12.
Val –
15.7.1 NONESSENTIAL AMINO ACIDS
Many of the nonessential amino acids can be obtained by reversing near-equilibrium reac-
tions that we have already considered or by the conversion from other amino acids. For
example, the synthesis of alanine, glutamate, and aspartate is accomplished by near-equi-
librium transamination reactions we have already discussed in the context of catabolism
(Figure 15.24). Glutamate is also formed as the product of the glutamate DH reaction. Also
shown are asparagine and glutamine formation, catalyzed by the corresponding synthetase.
Proline and arginine are synthesized from the common precursor glutamate (Figure 15.25).
After the formation of glutamate γ-semialdehyde, the routes diverge. Nonenzymatic cycliza-
tion to pyrroline 5-carboxylate leads to proline, whereas transamination of the semialdehyde
produces ornithine. Enzymes of the urea cycle are then employed to convert ornithine to
arginine.
Serine and glycine are linked not only in their catabolic pathways (as in the prior section),
but also in their biosynthesis. The amino acids are synthesized from the glycolytic inter-
mediate 3-P-glycerate (Figure 15.26). First, 3-P-glycerate is reduced to 3-P-hydroxypyruvate,
catalyzed by a dehydrogenase. Transamination produces P-serine, which is then dephos-
phorylated to serine. Serine is converted to glycine in a near-equilibrium reaction catalyzed
by serine hydroxymethyltransferase. Thus, with the availability of excess glycine, this enzy-
matic step alone can produce serine.
15.7.2 ESSENTIAL AMINO ACIDS
Roughly half of the amino acids required for protein synthesis must be obtained in the diet.
In some cases, at least a portion of the essential amino acid can be obtained from the metab-
olism of other amino acids. For example, in the degradation of methionine (Figure 15.22),
we found one of its products was cysteine. Similarly, tyrosine can be synthesized from the
hydroxylation of phenylalanine (Figure 15.15). However, the de novo synthesis of phenyl-
alanine and the other aromatic amino acids requires a separate pathway that exists only in
auxotrophic organisms. We will consider them next in more detail as an example of more
extensive essential amino acid biosynthesis.
15.7.3 AROMATIC AMINO ACID BIOSYNTHESIS
The aromatic amino acids tryptophan, phenylalanine, and tyrosine are formed in the cho-
rismate pathway, named for the common intermediate. Some of the steps are illustrated
in Figure 15.27. The starting points are phosphoenolpyruvate (PEP) from glycolysis and
FIGURE 15.24 Direct formation of amino acids. Single-step formation of alanine, glutamate,
aspartate, glutamine, and asparagine.
erythrose-4-phosphate (E4P) from the pentose phosphate shunt. The seven-carbon conden-
sation product is ultimately converted to shikimate, a ring structure that is the precursor to
the aromatic rings in the product amino acids. After a phosphorylation step, a second PEP
molecule condenses with the ring in a synthase-catalyzed step; this reaction is highlighted
as an enclosed box in the figure. It is significant as the site of action of a well-known broad-
spectrum plant growth inhibitor, popularly known as Roundup (Box 15.4).
15.8 NUCLEOTIDE METABOLISM
Most of the nitrogen in organisms resides in the amino acids of proteins. However, amino
acids (along with NH3) are extensively involved in the metabolism of other compounds. The
products most central to the needs of the cell are the nucleotides, which are necessary as
cofactors – such as ATP and NAD+ – and for DNA and RNA synthesis. We examine here
the synthesis of the pyrimidine and purine bases and aspects of the metabolism of these
compounds.
As an overview, consider the pathways outlined in Figure 15.28. In the left panel, pyrimi-
dine bases are shown to originate from the amino acids aspartate and glutamine, forming
the cyclic intermediate dihydroorotate. Dihydroorotate condenses with a ribose molecule
that has a 5′-phosphate and a 1′-pyrophosphate, phosphoribosyl pyrophosphate (PRPP). The
product of the pyrimidine pathway is uracil monophosphate (UMP). The purine pathway
shown on the right panel of Figure 15.28 starts with the PRPP, and the pyrophosphate group
is displaced by the nitrogen of glutamine, forming the intermediate P-ribosamine. The rest
of the purine ring is built on that nitrogen, using inputs from 1C metabolism in the form of
384 15.8 Nucleotide Metabolism
FIGURE 15.25 Arginine and proline biosynthesis. Following conversion of glutamate to the
semialdehyde, one branch forms the cyclic product proline, and the other the open-chain argi-
nine. The formation of arginine uses enzymes of the urea cycle.
FIGURE 15.26 Serine and glycine biosynthesis. The glycolytic intermediate P-glycerate leads to serine formation in three
steps. The serine can then be converted to glycine in a reaction catalyzed by serine hydroxymethyltransferase (HMT).
N10-formyl-THF, nitrogen from glutamine, carbon from CO2, and the incorporation of an
entire glycine molecule. The product of the purine pathway is inosine monophosphate (IMP).
15.8.1 PYRIMIDINE SYNTHESIS
The complete pathway for the formation of UMP is shown in Figure 15.29. The formation of
carbamoyl-P is catalyzed by carbamoyl-P synthetase II (CPS II). This reaction is similar to
CPS I of the urea cycle. For the CPS II reaction, glutamine is converted to ammonia on the
enzyme surface, and the reaction then proceeds in the same way as CPS I. Whereas CPS
I is localized exclusively in the mitochondrial matrix, CPS II is exclusively cytosolic. The
next step, catalyzed by aspartate transcarbamoylase, is the condensation of CAP with aspar-
tate, producing carbamoyl-aspartate. Dehydration of carbamoyl-aspartate follows, a reaction
FIGURE 15.27 Aromatic amino acid biosynthesis. After a condensation reaction to form
the heptulose analog, cyclization to shikimate and a phosphorylation step, a synthase and
subsequent dephosphorylation reaction produce chorismate, the precursor of the aromatic
amino acids. The synthase reaction (boxed) is the site of inhibition of the herbicide glyphosate
(Roundup; see Box 15.4).
catalyzed by dihydroorotase, leading to dihydroorotate. Dihydroorotase DH catalyzes the

subsequent oxidation of dihydroorotate to orotate. This reaction is similar to glycerol phos-
phate oxidase. It is embedded in the mitochondrial inner membrane, its active site faces the
cytosol, and electrons are donated to the mitochondrial respiratory chain by the lipid-soluble
mobile cofactor UQH2. Next, the ribose portion of PRPP attaches to orotate to produce oroti-
dine 5’-monophosphate (OMP). Finally, orotate decarboxylase catalyzes the removal of CO2
and the formation of UMP, the parent pyrimidine.
Box 15.4 The Chorismate Pathway and Weed Control
The chorismate pathway can be selectively inhibited by a compound developed by

Monsanto Chemical Company called glyphosate, popularly known as Roundup. The
inhibited step is the synthase reaction shown in the boxed area of Figure 15.27, which
is required for the synthesis of the three aromatic amino acids. Glyphosate inhibition is
competitive with the substrate PEP; the structures are shown in Figure B15.4. Glyphosate
is a phosphonate (note the P–C bond) and is broken down in the soil to the nontoxic prod-
ucts Pi, NH3, and HCO3−. The specificity of glyphosate is remarkable. Glyphosate appears
to have no effect on other reactions that use PEP, including the one at the beginning of
the chorismate pathway itself. Because the pathway does not exist in animals – the very
reason the amino acids are classified as essential – there is little direct human toxicity.
However, with the increased use of glyphosate, harmful effects have been discovered in
more species, including humans. Direct inhibition of the microbiome of bees, for exam-
ple, has endangered an already vulnerable species. The safety record of glyphosate is
thus less clear than it once was.
Glyphosate kills virtually all plants, not just weeds. It can nonetheless be simultane-
ously used with genetically modified crops (and, conveniently enough, also supplied by
Monsanto) with an altered synthase that does not bind glyphosate. As a further concern,
some weeds have evolved to become resistant to glyphosate.
FIGURE B15.4 Structures of glyphosate and PEP.
UMP is a substrate for nucleotide kinases, which use ATP as the donor to form first UDP
and subsequently, UTP. UTP is a substrate of the CTP synthetase reaction (Figure 15.30) in
which the nitrogen of glutamine is incorporated into the product.
15.8.2 PYRIMIDINE DEGRADATION
The breakdown of pyrimidine nucleotides starting from CMP and UMP is illustrated in
Figure 15.31. Dephosphorylation of CMP yields cytidine; deamination of this intermediate
produces uridine. UMP directly forms uridine by dephosphorylation. Uridine phosphory-
lase, which catalyzes the next reaction, has a mechanism similar to glycogen phosphorylase
(Chapter 13). Inorganic phosphate (Pi) adds to an intermediate ribose carbocation at Cl, pro-
ducing uracil and ribose-1-P. A later intermediate of pyrimidine degradation is β-alanine,
most of which is converted to succinyl-CoA and subsequently to glucose. However, some
β-alanine can be used in the synthesis of CoA itself, so pyrimidine degradation is connected
to the formation of this cofactor.
15.8.3 PURINE SYNTHESIS
In the overview of nucleotide synthesis (Figure 15.28), it is evident that purine biosynthesis
builds the nucleotide, starting with a sugar phosphate. In contrast, pyrimidine biosynthesis,
as we have just seen, forms the nucleoside first.
FIGURE 15.28 Overview of nucleotide metabolism. Pyrimidines (left side) form an aromatic
ring to which ribose is added. Purines (right side) start with a ribose scaffold and, after several
steps, lead to the finished nucleotide.
The first reaction of the sugar phosphate – PRPP – is the displacement of the pyrophos-
phate at the 1-OH group of the ribose by nitrogen of glutamine (Figure 15.32). In subsequent
steps, the ribose phosphate portion is symbolized as R, as shown in the figure. The enzymes
catalyzing this step, and the ensuing reactions are listed in Table 15.2.
The formation of IMP from P-ribosamine is illustrated as Figure 15.33. A synthetase cata-
lyzes incorporation of a complete glycine molecule (reaction (b)). The enzyme mechanism
involves an acid phosphate intermediate, evident from the splitting of ATP to ADP and Pi in
the overall reaction. Reaction (c) incorporates a formyl group onto the free amine, another
example of one-carbon metabolism. The product of reaction (c) is FGAR (formylglycinamide
ribonucleotide), which is converted to FGAM (formylglycinamide ribonucleotide) by incor-
poration of another nitrogen from glutamine in reaction (d). Ring formation (reaction (e))
requires yet another utilization of ATP bond energy, producing AIR (aminoimidazole ribo-
nucleotide). A biotin-dependent decarboxylation step (f) produces CAIR (carboxyamino-
imidazole ribonucleotide). The next two reactions – (g) and (h) – incorporate the nitrogen
of aspartate, analogous to arginine formation in the urea cycle (Section 15.4). In step (g),
FIGURE 15.29 UMP biosynthesis.
FIGURE 15.30 CTP synthetase reaction.

FIGURE 15.31 Pyrimidine degradation. CMP and UMP breakdown converge at uridine, ulti-
mately forming the intermediates CoA and succinyl-CoA.
FIGURE 15.32 Purine biosynthesis: first step. The formation of the amine sugar phosphate
from PRPP is abbreviated R-NH2 for the remaining stages, illustrated in Figure 15.33.
TABLE 15.2 Enzymes of Purine Metabolism

Step Enzyme
a Aminophosphoribosyl transferase
b GAR synthetase
c GAR transformylase
d FGAM synthetase
e AIR synthetase
f AIR carboxylase
g SAICAR synthetase
h Adenylosuccinate lyase
i AICAR transformylase
j IMP cyclohydrolase
FIGURE 15.33 Purine biosynthesis pathway to IMP. Enzymes catalyzing purine biosynthesis are listed in Table 15.2, corre-
sponding to the numbers shown in the figure.
aspartate condenses with the carboxyl group of CAIR, forming the succinylamide derivative
SAICAR (succinylamide carboxylaminoimidazole ribonucleotide). In step (h), fumarate is
released, and the nitrogen atom is incorporated into the growing nucleotide, forming AICAR
(aminocarboxamide ribonucleotide). Finally, step (i) incorporates the last carbon from N10 –
formyl-THF, and step (j) is a dehydration reaction that closes the ring, producing IMP.
The formation of GMP and AMP both involve incorporating nitrogen into the purine ring
of IMP, using reactions similar to those we have just encountered in the biosynthesis of IMP
itself except for reaction (1). The first step in the pathway for GMP formation (Figure 15.34,
FIGURE 15.34 GMP and AMP formation from IMP
FIGURE 15.35 Mechanism of IMP dehydrogenase
upper branch) involves an oxidation and incorporation of oxygen. This mechanism is very
similar to that of glyceraldehyde phosphate dehydrogenase (Chapter 9), in that it involves
covalent incorporation of the substrate through a sulfur atom of cysteine. The mechanism
(Figure 15.35) shows this similarity. IMP binds the enzyme in the first step. Next, NAD+
binds, abstracts a hydride, and is released as NADH. The enzyme-bound nucleotide is
released as a water molecule displaces the sulfur bond, thereby incorporating the second
oxygen into the purine ring to form XMP. XMP is converted to GMP in a reaction catalyzed
by a synthetase, similar to those of the purine pathway itself.
The lower branch of Figure 15.34 shows the two steps in the conversion of IMP into AMP.
These are also similar to the reactions of the urea cycle and of the IMP synthesis pathway.
First, the aspartate nitrogen condenses with the IMP to form the succinyl derivative of IMP,
followed by the removal of the carbon fragment as fumarate, forming AMP.
As was the case with the pyrimidine nucleotides, an array of kinases catalyze the con-
version of the pyridine monophosphates into di- and tri-phosphonucleotides. Furthermore,
nucleoside diphosphate kinase can catalyze the conversion of any of the diphosphates to
triphosphates using ATP as the other substrate.
15.8.4 PURINE DEGRADATION
The degradation of purines is a more active route than the pyrimidines because purines exist
in greater concentrations in cells and are used in more metabolic pathways. Adenine nucleo-
tides are responsible for most phosphate-bond energy transfers, so it is fitting that they are
present at 10–fold the concentration of the next major nucleotide, the guanine nucleotides.
The end product of purine nucleotide breakdown in mammalian systems is uric acid.
The pathway is shown in Figure 15.36. AMP can be either deaminated through the AMP
deaminase reaction or dephosphorylated through the nucleotidase reaction. For example,
FIGURE 15.36 Purine degradation. The scheme shows alternative pathways for AMP break-
down that are typically limited to separate cells. Purines ultimately form hypoxanthine and xan-
thine, converted by xanthine oxidase to uric acid, the excreted end product.
muscle cells have mostly the deaminase pathway, whereas T-lymphocytes have mostly the
nucleotidase pathway. Both of these routes converge on inosine by a further dephosphoryla-
tion or deamination, as indicated in the figure. Next, the ribose portion is removed through a
phosphorylase reaction of the type described above for pyrimidine catabolism (Figure 15.31);
the product is hypoxanthine. Xanthine oxidase catalyzes the oxygenation of hypoxanthine
to xanthine, as well as the further oxidation of xanthine to uric acid, which is excreted in
humans. GMP is catabolized in a similar way, through dephosphorylation, a phosphorylase
reaction, and deamination to xanthine, which merges with the AMP degradation pathway.
Xanthine oxidase is thus involved in the degradation of both adenine and guanine nucleo-
tides. Like the aconitase reaction of the Krebs cycle (Chapter 10), xanthine oxidase catalyzes
two sequential reactions. The reaction mechanism is complex: the oxygen atom inserted into
the ring arises from a water molecule bound to a molybdenum ion. Electrons removed from
the substrates are passed first to a Fe/S cluster, then to a FAD prosthetic group, and finally to
O2, leading to reaction oxygen species as coproducts, oxygen radicals, and hydrogen perox-
ide. These products are very reactive and potentially harmful to cells. They are inactivated by
enzyme systems devoted to reactive oxygen species in general. A further discussion of the
system is presented in the Appendix.
Uric acid is poorly soluble in water, so when excessive amounts accumulate in humans, it
can precipitate in joints, resulting in gout. Excess production is one cause, and this can be
treated by inhibiting xanthine oxidase with allopurinol. A second treatment strategy is to
block kidney reabsorption with probenecid. The structures of these compounds are shown
in Figure 15.37. Uric acid is the principal nitrogen end product in birds. The white semisolid
precipitate involves far less water, a weight-saving advantage for avian flight.
FIGURE 15.37 Allopurinol and probenecid: gout treatments.
15.8.5 SALVAGE REACTIONS
Not all of the purine nucleotides that are broken down are excreted as uric acid. In many
cases, they react with PRPP, using one of two enzymatic reactions to regenerate nucleotides.
The first of these is adenosine-P-ribosyl transferase:

(15.6) Adeno sin e + PRPP ® AMP + PPi
The second, hypoxanthine-guanosine transferase, uses guanosine or hypoxanthine as

substrate:

(15.7) Guano sin e + PRPP ® GMP + PPi

(15.8) Hypoxanthine + PRPP ® IMP + PPi
The salvage pathway’s importance is evident in the pathology of Lesch–Nyhan syndrome, the
result of a deficiency in hypoxanthine-guanine transferase. Without this salvage pathway,
purine breakdown products accumulate, leading to gout. Other symptoms of the disease,
such as mental defects, are less obviously related to the known enzyme deficiency.
Cells can take advantage of the salvage pathway in normal metabolism to bypass the far
more extensive de novo synthesis pathway. Re-using nucleotides represents a significant
energy saving for cells.
15.8.6 PURINE NUCLEOTIDE REGULATION
Allosteric control by adenine and guanine nucleotides was demonstrated for isolated enzyme
preparations in the late 1950s. Subsequent investigation focusing on microorganisms estab-
lished long-term (genetic) control for several of the steps of the pathway. However, compa-
rable control of de novo purine biosynthesis for animal species is not as well established. It
is unlikely to be the same; for example, ATP concentrations are generally unchanged during
most metabolic states of mammalian cells. Hence, ATP is not itself a metabolic regulator.
Free ADP and AMP concentrations fluctuate, but on a far shorter time scale than that which
would be appropriate for the control of nucleotide biosynthesis. Alterations in other nucleo-
tide concentrations are far less well-known.
The salvage pathways of purine biosynthesis are regulated in part by the concentration
of PRPP. In birds, which have extensive flows through a slightly modified purine biosyn-
thesis pathway to synthesize and excrete uric acid, increased synthesis of rate-limiting
enzymes, particularly xanthine oxidase and PRPP amidotransferase, are critical regulatory
steps.
15.8.7 PURINE NUCLEOTIDE CYCLE
A pathway specifically involving AMP is the purine nucleotide cycle. This cyclic route
involves just three reactions, one of which is the deamination of AMP to IMP that we have
already examined in the context of purine degradation. The cycle (Figure 15.38) regenerates
AMP, using the nitrogen of aspartate, through a condensation reaction followed by a split-
ting reaction with the release of fumarate. This pathway’s enzymes are found in exception-
ally high concentration in muscle, which uses fumarate as an anaplerotic reaction (filling
reaction) for the Krebs cycle. Muscle has little pyruvate carboxylase, which other cells use to
convert pyruvate to oxaloacetate and maintain levels of Krebs cycle intermediates.
As shown in Figure 15.39, the aspartate condensation reaction followed by fumarate
release is a common theme, present in the urea cycle, in purine biosynthesis, and in the
purine nucleotide cycle. A variation on this theme is the transfer of the N atom of lysine to
glutamate by the intermediate formation of saccharopine (Figure 15.14).
15.8.8 DEOXYNUCLEOTIDE FORMATION
Deoxynucleotides are formed from the corresponding nucleoside diphosphate (NDP) in

the reaction catalyzed by ribonucleotide reductase (Figure 15.40). The reaction shown in
Figure 15.40 is incomplete because the enzyme is left in an oxidized form. Regeneration of
the reduced form of ribonucleotide reductase involves two other proteins in an electron-
transfer chain, shown in Figure 15.41. First, thioredoxin reductase accepts a hydride from
NADPH, transfers the electrons to a bound FAD, and then to the disulfide of thioredoxin,
leaving that protein in the sulfhydryl form (i.e., reduced). Ribonucleotide reductase accepts
electrons from thioredoxin. The action of thioredoxin reductase on thioredoxin is closely
analogous to two other reactions in cells. One of these is the last enzyme of the dehydroge-
nase complexes (such as the pyruvate dehydrogenase complex), lipoamide dehydrogenase.
The other is glutathione reductase (Figure 15.42), which consumes NADPH in order to pro-
duce reduced glutathione from oxidized glutathione. Reduced glutathione reacts with many
intracellular proteins to maintain them in the sulfhydryl form as part of the antioxidant
system in cells. The common theme of these reactions is the passing of electrons between an
enzyme sulfhydryl, FAD, and a nicotinamide nucleotide (NAD+ or NADP+).
FIGURE 15.38 Purine nucleotide cycle. Cycling between these purine nucleotides converts
aspartate to fumarate, which is anapleurotic for the Krebs cycle in muscle.
FIGURE 15.39 Three instances of aspartate N incorporation. The conversion of aspartate to

fumarate in two steps is a similar metabolic route in different pathways of nitrogen metabolism.
FIGURE 15.40 Ribonucleotide reductase.
FIGURE 15.41 Electron flow through three proteins for dNTP formation. The scheme shows
the route of NADPH-dependent reduction of the ribose sugar to produce deoxynucleotides.
15.8.9 A UNIQUE METHYLATION TO FORM dTMP
Thymidine is required for the formation of DNA. It is formed through a methylation reac-
tion that is unique in 1C metabolism. The methylation is catalyzed by thymidylate synthase,
as illustrated in Figure 15.43 (top panel). The methyl donor is N5,N10-methylene-THF; the
cofactor also contributes a pair of electrons into the product deoxythymidine 5’-monophos-
phate (dTMP). The coproduct, dihydrofolate (DHF), is correspondingly oxidized. In all other
FIGURE 15.42 Glutathione reductase in protein sulfhydryl reduction. Glutathione (top panel,
in reduced form) is utilized to maintain reduced protein sulfhydryl groups. The reducing equiva-
lents are supplied by NADPH (bottom panel).
FIGURE 15.43 dTMP Formation. The formation of the DNA-specific nucleotide dTMP uses
reductive methylation (top panel). Restoration of the methyl donor requires NADPH input and
methylation of THF (bottom panel).
one-carbon reactions of folate, the redox state of the folate itself is unchanged. Regeneration
of THF from DHF is shown in the bottom panel of Figure 15.43, along with the essential
portions of the folate structures. The enzyme DHF reductase catalyzes the addition of a pair
of electrons across the double bond containing the N5 of DHF. Subsequently, THF can reac-
quire the methyl group through the serine hydroxymethyl transferase reaction as mentioned
previously in the discussion of amino acid catabolism. The DHF reductase step can be selec-
tively inhibited by compounds such as methotrexate
H2N N N H
CH3
N N
N
H
NH2 N H COOH
C
O H2C
CH2
COOH
Methotrexate
which starves DNA synthesis of deoxynucleotides. This drug was one of the first used in
cancer therapy, preferentially killing rapidly-dividing cells. Methotrexate is also used in
the treatment of psoriasis, a condition of excessive skin cell division. An entirely different
mechanism of action for the drug underlies its use as an anti-inflammatory agent (Box 15.5:
Methotrexate and Rheumatoid Arthritis).
Box 15.5 Methotrexate and Rheumatoid Arthritis
Methotrexate was synthesized in the 1940s as a drug to inhibit DHF reductase. However,
it soon became apparent that the drug was also useful in treating rheumatoid arthritis, an
inflammation of the joints. Initially, the hypothesis was that methotrexate acts as it does
on cancer cells, preventing multiplication of lymphocytes that produce inflammatory
cytokines. However, subsequent studies found that the dose of methotrexate effective
for arthritis is much less than that needed as an anticancer drug; clearly, the mechanism
must be different.
The other site of action for methotrexate is a step in the purine biosynthesis pathway
that uses N10 -formyl-THF as a methyl donor to AICAR (Figure B15.5). AICAR accumulation
inhibits AMP deaminase and adenosine deaminase, both of which lead to adenosine
accumulation. Adenosine binds extracellular receptors on immune cells that attenuate
the secretion of cytokines, thus alleviating arthritis. Caffeine, another inhibitor of ade-
nosine deaminase, may also have anti-inflammatory activity. Metformin, the common
diabetes drug, is also anti-inflammatory. While this may be due to its activation of AMPK
(Chapter 13), the drug has also been shown to inhibit AMP deaminase and adenosine
deaminase.
N10-formyl-THF
PRPP AICAR FAICAR AMP

- Pi
THF
Adenosine - NH3
methotrexate
- NH3
AICAR
IMP
Inosine
FIGURE B15.5 Mechanism of methotrexate suppression of inflammation. The route shown

is a condensation of the pathways presented as Figures 33, 34, and 36. Methotrexate inhib-
its 1C transfer to AICAR (step (i) of Figure 15.33), as the drug is a structural analog of folate.
The resulting accumulation of AICAR inhibits deamination reactions of AMP and adenosine,
leading to adenosine accumulation, which suppresses inflammatory cytokine production by
lymphocytes.
398 15.9 Other Nitrogen Pathways
FIGURE 15.44 Structural similarities in flavins, biopterins, and folates.
With the two reduction states of folate, we can now appreciate the strong similar-
ity between three bound cofactors: flavins, biopterins, and folates (Figure 15.44). The oxi-
dized forms are on the left side of Figure 15.44. Both flavins and biopterins allow one- and
two-electron transfers; they can react, for example, with metals or hydrides like NADH.
Figure 15.44 illustrates the chemical similarity in the pterin rings of biopterins and folates,
indicative of similar origins.
15.9 OTHER NITROGEN PATHWAYS
A large number of other routes exist in nitrogen metabolism. These are quantitatively more
minor than those already described, and their biological distribution is more limited. In the
plant and microbial world, the number of unusual end products that contain nitrogen is vast;
they are known collectively as the alkaloids. Their role in the organism that produces them
is not always obvious. In many cases, they discourage animals from eating them because
the compounds are bitter-tasting. In some cases, alkaloids are important drugs, such as the
analgesic morphine, isolated from the poppy flower.
HO
H N
CH3
HO
Morphine
Some neurotransmitters and similar signal molecules are biosynthesized from amino acids
(Figure 15.45). For example, decarboxylation of histidine produces histamine, which is
released from white blood cells and is partly responsible for allergy symptoms. Hydroxylation
FIGURE 15.45 Signal molecules derived from amino acids.
FIGURE 15.46 Nitric oxide synthesis.
and decarboxylation convert tryptophan into serotonin (5-hydroxytryptamine), the neu-

rotransmitter introduced in Section 15.3.
Finally, we consider nitric oxide production (NO), which may be the chemically simplest
cell modulator. The pathway for NO formation is the oxidation of arginine (Figure 15.46).
The enzyme catalyzing this step is nitric oxide synthetase. Its reaction mechanism is surpris-
ingly complex, involving the bound cofactors tetrahydrobiopterin and flavin mononucleotide
(FMN). In the body, NO synthase is found in the lining cells of the blood vessels (endothelia),
and the target of NO is the smooth muscle, where it causes relaxation, and thus blood ves-
sel dilation. Direct formation of NO can also be achieved by ingesting nitroglycerin, which
nonenzymatically releases NO.
SUMMARY
The global nitrogen cycle connects atmospheric N2 with NH3 and amino acids. Most bio-
logical nitrogen metabolism involves amino acids and NH3. Two near-equilibrium reactions,
glutamate DH and transaminases, are prominent in the synthesis and degradation of amino
acids and nucleotide metabolism. The urea cycle is the mammalian pathway by which amino
acids and NH3 are converted into urea in the liver. This route is connected to gluconeogenesis,
a pathway followed by the fumarate released from the urea cycle. Mammals excrete urea as
the major nitrogen end product. Breakdown of amino acids leads to glucose formation (glu-
cogenic), acetyl-CoA (ketogenic), or both. Some amino acid degradation pathways involve
just a single step, such as converting alanine to pyruvate by transamination. The breakdown
of serine, glycine, and methionine is linked to one-carbon (1C) metabolism, involving the
transfer molecules THF (tetrahydrofolate) and SAM (S-adenosyl methionine). In turn, 1C
transfer can contribute to the synthesis of other amino acids or other cellular structures.
The biosynthetic pathways of amino acids in humans can be divided into two groups: nones-
sential and essential. Nonessential amino acids can be synthesized endogenously, whereas
essential amino acids require the ingestion of outside sources. For example, aspartate can be
formed from oxaloacetate by a transaminase reaction, so aspartate is a nonessential amino
acid. On the other hand, the aromatic amino acids, such as tryptophan, require dietary input.
In the biosynthesis of pyrimidine nucleotides, a relatively short pathway leads to the con-
struction of the pyrimidine base, followed by the addition of a ribose sugar to form UMP.
The purine nucleotides are synthesized by a more elaborate pathway, but they begin with
the ribose phosphate sugar and build the nucleotide on that scaffold. P-Ribose-PP also reacts
with nucleosides to reform nucleotides in reactions collectively called the salvage pathway,
saving the energetic cost of complete biosynthesis. The degradation of purines proceeds by
deamination and dephosphorylation to an oxidized ring form, uric acid, which is excreted.
The deoxynucleotides are formed from the corresponding nucleotide diphosphate, using a
series of electron transfer reactions involving a disulfide protein exchange with thioredoxin
and the enzyme thioredoxin reductase. The formation of dTMP – required for DNA – is
unique. A methylated form of THF provides the methyl group that converts the uracil to a
thymidyl nucleotide. The resulting cofactor – DHF (dihydrofolate) is not only demethylated
but oxidized. Regeneration of THF requires a reduction catalyzed by the enzyme dihydro-
folate reductase. Many other, more specialized reactions occur in nitrogen formation, most
of which produce specific end products in plants or bacteria collectively known as alkaloids.
One significant reaction in mammals is the conversion of arginine to nitric oxide, which
causes smooth muscle relaxation in blood vessels.
REVIEW QUESTIONS
1. Many fish secrete ammonia as their nitrogen waste, but some secrete allantoin, which
is a further degradation product of uric acid. The formation of allantoin, a more water-
soluble molecule, is catalyzed by allantoinase. Describe how this enzyme might be
used as a drug therapy in humans.
2. Ammonia toxicity in humans may result from liver damage, but the most severe
effects are found in the brain. Describe the nitrogen metabolism pathways involved.
3. Explain how two key portions of the pyridoxal phosphate molecule – the carbonyl
group and the phosphate – are used in transaminase reactions and in phosphorylase
reactions.
4. How are the biopterin and flavin coenzymes similar?
5. SAM is sold commercially as “SAM-e” as a natural product that can supposedly treat
liver disease and depression. Yet, no controlled studies have shown such benefits.
Why would SAM appear from the written pathway to have these actions? Would
ingested SAM enter cells?
6. How would urea synthesis from glycine differ from urea synthesis from alanine?
7. Why are branched-chain amino acids considered metabolically as a unit?
8. After the formation of nitric oxide, what is the fate of the rest of the arginine molecule?
9. Why isn’t the urea cycle, starting with arginase, a possible pathway for arginine catab-
olism? How can the urea cycle be considered as part of the pathway for arginine
anabolism?
10. How can adenosine be formed from either AMP deaminase or adenosine deaminase?
11. Why aren’t anticancer drugs developed to inhibit pyrimidine metabolism?
CHAPTER 15 ADDENDUM: NITROGEN DISPOSAL
Humans excrete nitrogen through the kidneys in three forms: creatinine (about 5%), uric acid
(about 25%), and urea (about 65%); only minor amounts of free ammonia are found in the
final urine. Accordingly, we are considered ureotelic. The preponderance of secretion prod-
uct depends principally upon the water environment of the organism. Fish, which can dilute
ammonia extensively so that it is no longer toxic, are ammoniotelic, energetically the cheap-
est means of nitrogen excretion. Adult frogs are ureogenic, but tadpoles are ammoniotelic;
in this case, the transformation involves induction of urea cycle enzymes. Birds, minimizing
their weight for flight, use the least water, and excrete uric acid (uricotelic).
Here we detail three situations in which excretion patterns are unusual. The first is a
biological adaptation of fish in highly alkaline waters. The second is a distinction between
alligators and crocodiles in nitrogen elimination. The third is a case of liver disease that alters
normal creatinine excretion.
THE TILAPIA OF LAKE MAGADI
In Kenya, there is an alkaline lake (Magadi), with a pH of 10. Investigators from the University
of Nairobi in 1989 reported a tilapia species that thrives in this lake through its ability to syn-
thesize urea as its exclusive nitrogen disposal molecule. As a control, a distinct tilapia species
from another lake in Kenya having a pH of 7.1, forms only ammonia, and dies within an hour
of being placed in Lake Magadi.
The alkaline lake tilapia have a urea cycle similar to the one described in this chapter, with
one exception: there is no direct incorporation of ammonia in carbamoyl-P. This role is taken
over by a mitochondrial glutamine-dependent reaction, CPS III. The liver of Magadi tilapia
also has a cytosolic CPS II, for normal functioning in pyrimidine synthesis.
If we step back from this remarkable discovery, we can appreciate why ammonia secretion
requires copious water to dilute it and a relatively low pH. Direct ammonia secretion requires
that the pH of the kidney epithelial cytosol be significantly higher than that of the extracel-
lular fluid – which will carry the ammonia into tubule lumen, and ultimately the final urine.
In the situation where the extracellular pH is very high – that is, much greater than the
intracellular pH of 7.1 – there will be no capture of the ammonia nitrogen as ammonium ion,
and thus, little export.
THE ALLIGATOR AND THE CROCODILE
These two crocodilian species have a divergence in nitrogen elimination that reflects their
habitat. Alligators are mostly freshwater dwellers, and, like fish, they excrete ammonia.
Crocodiles can inhabit salt water due to their ability to remove Na and Cl ions through a
gland on the surface of the tongue. To conserve their internal water, crocodiles synthesize
uric acid as their major nitrogen excretion product. It is also possible for crocodiles to inhabit
a freshwater environment, in which case the organism becomes ammoniotelic. Thus, the
purpose of uric acid formation by the crocodile is to conserve water, a distinct biological
reason from the weight requirement of birds.
LOW CREATININE AND LIVER FAILURE
Creatinine clearance has long been a standard means of determining kidney filtration. It is
the least invasive measurement: only blood and urine creatinine levels are required for the
estimate. Of course, it is also known that it suffers from the fact that creatinine is not only
filtered and excreted from the kidney, but also secreted; that is, added back to the tubule
lumen after the filtration event. Thus, the method overestimates the filtration rate somewhat.
Serum levels of creatinine are also an indication of deterioration of the liver, such as in
cirrhosis. Understanding the reason behind this requires us to examine the metabolism of
creatinine more closely.
The pathway of creatinine formation is illustrated in Figure A15.1. Reaction (1) is cata-
lyzed by glycine amidinotransferase, a kidney enzyme. The ornithine formed can be recon-
verted to arginine through the ornithine transcarbamoylase, found in the kidney but utilized
for arginine resynthesis – not for the eventual formation of urea. The other product of reac-
tion (1) is guanidinoacetate, which exits the kidney and enters liver cells, where it undergoes
reaction and (2), a methyltransferase using S-adenosylmethionine (SAM). This reaction is
402 Key Terms
FIGURE A15.1 Formation of creatine, creatine-P, and creatinine.
prodigious, responsible for over half of the SAM methylation reactions of liver. The product,
creatine, exits the liver and enters tissues such as muscle and brain which utilize creatine as a
buffer of high-energy phosphoryl groups. CPK catalyzes the formation of creatine-P (reaction
(3)) in muscle and brain; most of the creatine-P is utilized for the reverse direction of reaction
(3) under times of high energy demand to maintain the constancy of ATP concentration. A
small amount of creatine-P is nonenzymatically converted to creatinine through the non-
enzymatic reaction (4). While it had earlier been postulated that creatine itself could form
creatinine, this is not the case, as demonstrated by chemical kinetic studies.
Thus, it is clear why liver diseases, such as cirrhosis, lead to diminished blood levels of
creatinine: the liver is part of the multi-organ pathway of creatinine formation. Thus the low
level of creatinine under these circumstances explains the diminished level of creatinine
in the blood, as well as the inability to use creatinine clearance as a measure of kidney flow
(GFR) in cirrhosis.
KEY TERMS
alkaloids
allopurinol
ammoniotelic
anaerobes.
assimilation
biopterin
cobalamin
corrosive
denitrification
essential amino acid
glucogenic
gout
intrinsic factor
ketogenic
leghemoglobin
maple syrup urine disease
methionine cycle
nitrification
nitrogen cycle
nitrogen fixation
nitrogenase
nonessential amino acid
ping-pong mechanism
probenecid
reactive oxygen species
S–adenosylmethionine
semialdehyde
sequential mechanism
thioredoxin reductase
ureido
ureotelic
uricotelic
vitamin B12
REFERENCES
G.W. Allen, P. Haake. Mechanism of Phosphorylation by N'-Phosphorocreatine. Concurrent Formation
of Creatine and Creatinine. J. Am. Chem. Soc. 98 (1976) 4990–4996.
J.P.K. Gill, N. Sethi, A. Mohan, S. Datta, M. Girdhar. Glyphosate Toxicity for Animals. Environ. Chem.
Lett. 16 (2018) 401–426.
Y.A. Kim, M.T. King, W.E. Teague, Jr., G.A. Rufo, Jr., R.L. Veech, J.V. Passonneau. Regulation of the
Purine Salvage Pathway in Rat Liver. Am. J. Physiol. 262 (1992) E344–352.
E.V.S. Motta, K. Raymann, N.A. Moran. Glyphosate Perturbs the Gut Microbiota of Honey Bees. Proc.
Natl. Acad. Sci. USA 115 (2018) 10305–10310.
T. Nakagawa, H. Hu, S. Zharikov, K.R. Tuttle, R.A. Short, O. Glushakova, X. Ouyang, D.I. Feig, E.R.
Block, J. Herrera-Acosta, J.M. Patel, R.J. Johnson. A Causal Role for Uric Acid in Fructose-
Induced Metabolic Syndrome. Am. J. Physiol. Renal Physiol. 290 (2006) F625–631.
R.J. Rolfes. Regulation of Purine Nucleotide Biosynthesis: In Yeast and Beyond. Biochem. Soc. Trans.
34 (2006) 786–790.
Nucleic Acids 16
1952, James Watson and Francis Crick revealed the structure of DNA in a concise single-
page paper in the journal Nature. It was the first announcement of the double helix and
showed how two complementary strands of DNA, noncovalently bound, spiraled around one
another in pleasing symmetry. The landmark study concluded with the observation that the
structure immediately suggests a means for DNA replication: as the strands separate, each
could serve as a template for a new daughter strand.
A few years later, Crick proposed the central dogma: “DNA makes RNA makes protein,” which
succinctly summarizes this chapter. A slight revision to this pathway followed the discovery that
some viruses have RNA as their genetic material. The viral RNA is first converted to DNA in the
host cell, required for replication. Subsequently, the central dogma pathway proceeds.
We will examine some of the biochemical underpinnings of the central dogma, starting
with the nucleic acids, DNA and RNA, the last biochemical polymers introduced in this
book. The study of nucleic acid and protein metabolism is the essence of molecular biology, a
term that blurs the lines between a biological and a chemical approach. In the present chap-
ter, we will consider chemical features of the nucleic acids, the processes of duplication of
DNA (replication), and the formation of RNA from DNA (transcription).
16.1 STRAND STRUCTURES OF THE NUCLEIC ACIDS
The individual strands of DNA and RNA have a similar structure (Figure 16.1). The DNA
polymer is linked by phosphodiester bonds between the deoxyribose moieties of the nucleo-
tides. These bonds are the only covalent linkages in the polymer, and they also define a direc-
tion for the strand. The first subunit shown has a free phosphate group at the 5' position;
accordingly, it is called the 5' end. Joining the 3' carbon of the first subunit to the 5' carbon
of the second unit via a phosphate linkage is repeated throughout the chain. DNA polymers
are the largest in nature; the human chromosome has 250 million base pairs. RNA shares a
similar backbone structure with DNA but is five orders of magnitude smaller and has ribose
sugars in place of the deoxyribose sugars of DNA.
Another distinction between DNA and RNA strands is the identity of the bases, as indi-
cated in Figure 16.1. DNA has four bases: adenine (A), guanine (G), cytosine (C), and thymine
(T). RNA also has four bases: three are the same as those in DNA (A, G, and C), but uracil
(U) is uniquely present in RNA.
Figure 16.2 shows a detailed structure of a segment of RNA, with the nucleotide bases U
and G shown. The nucleotides are the unique portion of each unit of the chain. Accordingly,
we use a shorthand to represent the nucleic acid strands. The position of the phosphate can
only be 3' or 5'. A lowercase p is written to the left of the base symbol to represent the 5' side:
. . . pUpG . . .
Intervening phosphates can be omitted so that a lengthy sequence such as:
pCGAAUGCC
unambiguously identifies a strand of RNA with a phosphorylated cytosine at the 5' end.
405
406 16.1 Strand Structures of the Nucleic Acids
FIGURE 16.1 Backbone strand structures of DNA and RNA.
FIGURE 16.2 RNA segment
When DNA strands are represented, the deoxyribose can be indicated by inserting a low-
ercase d:
pdGdTdTdCdAdCdG
but this is usually omitted so that this sequence becomes simply:
pGTTCACG
It is possible to confuse the strands if there are no thymine or uracil bases, but, in context,
the representation is usually unambiguous. Another alternative is to indicate the 5' and 3'
ends explicitly with numbers:
5'-GTTCACG-3'
Chapter 16 – Nucleic Acids 407
16.2 STRUCTURE OF THE DOUBLE HELIX
DNA has two strands joined by noncovalent forces. Figure 16.3 shows the sugar-phosphate
backbones of the two strands in a ladder-like structure in which the “rungs” consist of a
nucleotide base contributed by each strand. Each rung has a purine and pyrimidine hydro-
gen-bonded together so that they are all of uniform length. Invariably, a T always appears
opposite an A, and a G always appears opposite a C. Edwin Chargraff discovered this rule
prior to the discovery of the DNA structure: the total number of A bases equals that of T
bases, and the total number of G bases equals that of C bases. Thus, the rungs consist of
either AT pairs or GC pairs. Note that there are two hydrogen bonds in the AT pair and three
in the GC pair. Because of this rule of complementarity, the same information is present in
each half of the double helix. That is, once the sequence of one strand is known, the other can
be readily predicted. It also suggests a mechanism for replication: if the two strands separate,
each can serve as a new daughter strand template.
There is further detail to the DNA structure. The base pairs in the center, due to their
alignment and formation of hydrogen bonds, are planar and hydrophobic. They stack on top
of each other – somewhat offset – in three dimensions, as if they were flat rungs in a spiral
ladder, while the sugar-phosphate strands form the “sides” of the ladder, wrapped in a helix
(Figure 16.4). The helix has features in common with the protein α-helix introduced previ-
ously in this text. The two are compared in Figure 16.5. In each case, an inner hydrophobic
FIGURE 16.4 Three-

dimensional form of the
FIGURE 16.3 Two-dimensional view of DNA base pairing. DNA helix
408 16.2 Structure of the Double Helix
FIGURE 16.5 The DNA double helix and the α-helix of proteins. The core in both cases is
hydrophobic. DNA has a hydrophilic exterior, whereas the α-helix exterior may be either hydro-
phobic or hydrophilic, depending on the nature of the R-group side chains that extend to the
outside of the structure.
FIGURE 16.6 Attractive forces behind stacking interactions. (a) Two benzene rings model the
base-paired “rungs” of Figure 16.4. (b) Side view of the rings shows π–electron clouds above
and below each ring (with partial negative charge), leaving partial positive charge at the edges.
(c) Offsetting the rings maximizes the stabilization due to charge attractions.
core is internally hydrogen-bonded and cannot form hydrogen bonds to water. The exterior
of the α-helix can be hydrophilic or hydrophobic depending on the R groups of the individual
amino acids. DNA, however, has a strongly polar, negatively charged exterior due to the dis-
sociated phosphate groups that link the nucleotide subunits.
Helix formation in DNA is driven largely by interactions between the “ladder rungs” (i.e.,
the AT or GC base pairs). Each hydrogen-bonded base pair rung experiences an attractive
force between rungs above and below them. This attractive force is known as the stack-
ing interaction. The base pairs are flat, aromatic, and hydrophobic. To explore the basis
of the stacking interaction, we will turn to the benzene ring, a simple model compound.
Figure 16.6a depicts two benzene rings that illustrate the stacking interaction. The same pair
of benzene rings is shown in an edge-on view in Figure 16.6b, with the pi electron clouds
FIGURE 16.7 Major and minor grooves. Each flat “rung” of the DNA ladder has a larger gap or groove on one side (the major
groove) than the other (the minor groove). The relative sizes of the gaps depend upon the identity of the base pairs, providing
different binding sites for proteins that interact with DNA.
explicitly drawn above and below each ring. Note that the edges of each ring have a relative
positive charge. In Figure 16.6c, stability is achieved due to the offsetting of the rings, allow-
ing positive and negative portions to interact.
Like the aromatic benzene rings, each rung in the DNA ladder is offset from the one below
it. The three-dimensional structure forms as a spiral staircase. Hence, the stacking interac-
tion directs the offsets of base pairs that give rise to the helical nature of the DNA double
helix.
The positioning of the sugar residues of DNA is shown in Figure 16.7. Viewed from the
top of the helix, the sugar residues are arranged asymmetrically, producing a larger separa-
tion called the major groove and a smaller separation called the minor groove. The major
and minor grooves provide one level of discrimination for binding: different proteins fit into
the two grooves. Four distinct edges are pictured in Figure 16.7, resulting from differences
between AT and GC base pairs and from differences between the major and minor grooves.
Thus, a segment of DNA provides selectivity for proteins based upon the identity of the bases
and the distinct binding of proteins to the major or minor groove.
The unwinding of the DNA is required for its biological functions, such as replication
and transcription. Intrinsically, the AT pair is split more easily than GC, which is reflected
in melting curve experiments in which synthetic DNA strands containing only A and T are
410 16.3 Supercoiling
FIGURE 16.8 Melting curves for DNA. Naturally occurring DNA has a midpoint in the melting
curve (Tm) between poly-AT (low Tm) and poly-GC (high Tm). The results show experimentally
that GC pairs are more stable than AT pairs.
compared to synthetic strands containing only G and C (Figure 16.8). As the temperature
is increased, the DNA strands separate, which is indicated by increased absorption of ultra-
violet light. The midpoint of the sharp increase in dissociation (called the melting tempera-
ture) is much lower for AT-rich strands than for GC-rich strands.
AT-rich regions exist at DNA replication origins, suggesting that the hydrogen-bonded
bases in these segments are intrinsically easier to break. There are three hydrogen bonds
in the GC pair and just two in the AT pair, but hydrogen bond energy differences are not
the entire reason for the greater strength of the GC pairs. GC pairs also have greater stack-
ing interaction energy. As a result of the increased hydrogen bonding, the GC pair is more
planar, increasing the aromaticity of the dual ring system, accounting for the increase in the
stacking interaction.
16.3 SUPERCOILING
Like twisting a coiled phone cord, the DNA helix can exist in supercoils. When the number
of strand crossovers increases, the result is called a positive supercoil. Helices that have a
decreased number of crossovers have a negative supercoil. Enzymes that catalyze changes in
DNA supercoiling are called topoisomerases.
Bacteria contain two similar enzymes: topoisomerase I and topoisomerase II. The first
catalyzes the hydrolysis of one of the phosphodiester bonds in one chain, moves a DNA
strand through the opening, and then rejoins the chain. Hydrolysis of a phosphodiester bond
joining the backbone sugars of nucleic acids causes a breakage, commonly called cutting
(sometimes whimsically illustrated with miniature scissors). The mechanism of the reaction
involves two sequential nucleophilic substitutions, shown in Figure 16.9. A tyrosine hydroxyl
group of the enzyme displaces a portion of the strand, forming an enzyme-bound interme-
diate. Subsequently, the 5'-OH group of ribose attacks the same phosphate group to rejoin
the chain. Except for the change in topology (Figure 16.10), the product is the same as the
substrate.
Topoisomerase II cuts both strands, allows the movement of a separate piece of double
helix through, and then catalyzes resynthesis of the phosphodiester bond. In the course of
this reaction, ATP is converted to ADP and Pi, an indication that the process is energy-
dependent. The chemical mechanism for splitting and rejoining the sugar phosphate groups
is the same as topoisomerase I (Figure 16.9). Eukaryotic cells have similar enzyme activities,
although more topoisomerase isoforms exist.
FIGURE 16.9 Mechanism of topoisomerase I. The enzyme has an active tyrosine hydroxyl
group that attacks the phosphate “backbone” and temporarily binds one portion. Displacement
by the ribose OH of the transiently freed chain rejoins the DNA. The substrate and product are
chemically identical but topologically distinct.
FIGURE 16.10 Topology of the topoisomerase I reaction.
16.4 HISTONES
In eukaryotic cells, DNA strands bind basic proteins called histones, which exist in several
isoforms. A cluster of four histone dimers roughly forms a cylinder around which DNA is
wrapped, and yet another histone isoform binds a portion of the free DNA between the his-
tone clusters. The binding interaction is electrostatic: the positively-charged histones (having
excess basic amino acid side chains) attract the negative charges of the phosphate backbone
of DNA. Proteins bearing a net positive charge are rare in cells; the majority have a net nega-
tive charge at the pH of the cytosol (about 7.1). In addition to histones, the other positively
charged molecules that bind DNA are small basic molecules, the polyamines (Figure 16.11).
These molecules, found in both bacteria and eukaryotic cells, also bind the external phos-
phate groups of DNA through electrostatic attraction. A sharp rise in polyamine synthesis
accompanies cell division, coincident with DNA synthesis.
412 16.5 Replication
FIGURE 16.11 Polyamine structures.
1. DnaA oriC
2. DnaB
FIGURE 16.12 Location and sequence of initiation complex for replication in E. coli.
16.5 REPLICATION
The duplication of DNA – replication – involves separating the two strands of the molecule
and then copying each. This process is commonly divided into three stages: initiation, elon-
gation, and termination. The enzyme that catalyzes the synthesis of the new bases is DNA
polymerase, which incorporates nucleoside triphosphates (NTPs, where N = A, G, C, or T)
on a template starting from the 5' end. Thus, replication proceeds in the 5' → 3' direction.
The earliest studies of replication involved Escherichia coli (E. coli), for which most of the
mechanisms are best understood and are very similar to those in eukaryotes. We will note
some of the distinctions in the course of our discussion.
16.5.1 INITIATION
E. coli DNA is circular, but in resting states (i.e., when replication or transcription is not
occurring), it is supercoiled. As described in Section 16.3, topoisomerases catalyze the cleav-
age and rejoining of the phosphodiester bonds of the DNA backbone. A prominent topoi-
somerase in bacteria is known as DNA gyrase. Replication itself begins with the binding of
the protein DnaA to a specific 245-base pair (hp) region of the E. coli chromosome called
oriC, short for “origin of replication.” This region is AT-rich and intrinsically easier to unravel
(recall that GC pairs are more tightly bound than AT pairs). A complex of multiple copies of
DnaA bound to oriC serves as a binding site for another protein: the helicase enzyme DnaB
(Figure 16.12). Helicases catalyze the separation of the DNA strands to present templates
for replication. Two copies of the hexameric DnaB bind the DnaA:oriC complex, and DnaB
moves along a DNA strand. DnaB binds ATP, and the transfer of bond energy drives the
movement. Upon displacement along the strand, the enzyme releases ADP and Pi. The move-
ment of helicases along the surface of a DNA strand is an example of a molecular motor, like
the F1Fo ATP synthetase complex (Chapter 11).
16.5.2 REPLICATION FORK AND THE REPLISOME
Once the two strands of DNA are separated, a protein binding to a single strand of DNA
maintains the open structure, thus making it possible to initiate replication. This protein is
positively charged, and it selectively attaches to single strands through a central channel in
its tetrameric structure.
With the two strands of DNA separate, ensuing reactions add nucleic acids to both single
strands. The open “bubble” of separated strands is called the replication fork (Figure 16.13)
and is the site of the key reactions of the process. Because the strands run in opposite direc-
tions, each is copied in a distinct manner. One chain, called the leading strand, is extended
continuously in the 5' to 3' direction because its template starts with the 3' end. The other
chain, called the lagging strand, must be synthesized in the opposite direction in order to
use the same DNA polymerase. As illustrated in Figure 16.14, the lagging strand is synthe-
sized in short pieces, named Okazaki fragments, after the investigator who first discovered
them. The lagging strand must be formed in segments because of the directionality restric-
tion of nucleotide polymerases. While the leading strand always has a new template to act
upon as the DNA opens up, the lagging strand grows in the direction opposite to the replica-
tion fork; of necessity, it must terminate, and a new fragment must begin.
The synthesis of new DNA strands involves many proteins bound together in a complex
called the replisome. Both the leading and lagging strands contact the same complex because
FIGURE 16.13 The replication fork in prokaryotes and eukaryotes.
FIGURE 16.14 Leading and lagging strands in DNA replication.

414 16.5 Replication
FIGURE 16.15 The replisome. As the lagging strand loops back, both are copied at the same
rate. Several proteins (not shown) are required in this complex to unwind the DNA strands
(topoisomerase), assist the DNA polymerase (a primosome complex, containing helicase and
primase), and maintain portions of the lagging strand as a single chain (single-stranded bind-
ing protein).
the lagging strand loops back to engage the synthetic machinery (Figure 16.15). Accordingly,
the rate of synthesis of both strands is the same.
16.5.3 PRIMER FORMATION
The first reactions that add bases to single-stranded DNA are catalyzed by an RNA poly-
merase, known as primase. The enzyme binds first to DNA gyrase and several other proteins,
forming a complex called the primosome. The primase reaction product is a short chain of
RNA complementary to the DNA. The complementarity rules for DNA-RNA hybrids are
similar to those for DNA–DNA hybrids, except that RNA polymers use uracil in the place of
thymidine. The reaction itself is similar to most synthetic reactions of nucleotides and other
nucleophilic reactions involving phosphate groups. All nucleotides enter as triphosphates,
and they are joined through a nucleophilic displacement mechanism. The first nucleotide
retains the triphosphate at the 5' end of the sugar, and its 3'-hydroxyl group attacks the α–
phosphate of the next incoming nucleotide triphosphate. This process is repeated until about
11 base pairs have formed, at which point primase disengages from the chain. Nucleotide
polymerases generally engage the template for many rounds of synthesis before dissociating.
This activity is called processivity, distinct from of the more common enzyme mechanisms
in which a substrate is bound and released for each catalytic cycle.
Primer formation for the leading strand occurs just once in replicating DNA but multiple
times for the lagging strand. The lagging strand synthesis proceeds opposite the replication
fork, and new start sites are required as the duplex opens.
16.5.4 CREATING THE DOUBLE HELIX
With the primer sequence in place, the enzyme DNA polymerase can create new DNA
strands. DNA polymerase resembles RNA polymerase. In both cases, a nucleophilic attack
by the 3'-hydroxyl group of one end displaces pyrophosphate and form a new phosphodiester
bond. DNA polymerase uses dNTPs as substrates. In bacteria, the major isoform is DNA
polymerase III (named for its order of discovery). The leading strand forms a long uninter-
rupted chain of bases. In contrast, the lagging strand once again only synthesizes fragments,
each about 1000 nucleotides in length. DNA polymerase then dissociates, and a new section
of lagging strand undergoes another cycle of priming with RNA and extension with DNA.
After the replication events described are complete, the lagging strand consists of short
segments of RNA, followed by DNA, and then short interrupted single-strand segments. The
enzyme DNA polymerase I catalyzes the excision of the RNA as well as the synthesis of new
DNA to fill the gaps. Exonuclease, a hydrolase activity, catalyzes the removal of RNA. A gap
results in the DNA after the segments are completed by the action of polymerase III. DNA
ligase catalyzes the joining of phosphodiester bonds between these chains.
16.5.5 DISTINCTIVE FEATURES OF EUKARYOTIC REPLICATION
The process of DNA duplication in eukaryotes is similar to that of bacteria, although more
components are involved, and the details of the process remain less well understood. There
are, for example, nine DNA polymerases. Because the length of the DNA is far greater in
eukaryotes, it is not surprising to find that there are multiple initiation sites rather than just
one per chromosome, as in prokaryotes. More binding proteins are involved in forming these
replication complexes than in prokaryotes, and further controls exist to ensure that replica-
tion occurs only once in the cell cycle.
Perhaps the most striking distinction of eukaryotic DNA replication stems from the fact
that its DNA is not only more extensive, but it is arranged linearly rather than in the circu-
lar form of prokaryotes. With linear DNA, the lagging strand cannot be replicated entirely,
because the exposed DNA reaches a free end (the 3' end), and a new Okasaki fragment can-
not be initiated due to insufficient length. Thus, this unreplicated segment leaves a single 3'
overhang end, which requires a special system for its replication.
The sequence at these 3' ends (called telomeres; Latin, telos, meaning end) in vertebrate
chromosomes consists of thousands of repeats of the sequence TTAGGG. The enzyme
telomerase contains a complementary sequence as a cofactor and accordingly binds and
catalyzes the extension of the overhang. Upon completion, a small overhang remains, which
is bound by a protein complex, a process called capping. This largely prevents degradation of
the chromosomal telomeres, although there is some nuclease activity nonetheless. A short-
ening of chromosomes with age at the telomeres is a well-established phenomenon.
16.6 DNA REPAIR
Two major repair systems exist to ensure the fidelity of DNA reproduction. One is to correct
errors caused by inserting the incorrect base during normal replication: this is called mis-
match repair. The other is to fix the damage that occurs in DNA after it has been replicated,
an event strictly outside of the overall process of replication but relevant for the proper func-
tioning of DNA: excision repair.
16.6.1 MISMATCH REPAIR
Since mismatch repair occurs when the template is not faithfully replicated, the repair sys-
tem must determine which of the two strands is the parent and which is the daughter, with
the errant bases. The ambiguity is resolved as the parent strand is invariably methylated,
but the daughter strand is not – at least for some time after DNA synthesis. A newly formed
duplex is thus hemimethylated. In bacteria, mismatch repair begins when specific binding
proteins recognize the bulge of an unpaired or mismatched DNA, causing this segment to
form a loop. The protein complex slides along the DNA surface, increasing the loop, until the
methylation site – typically GATC – is encountered. The adenine is N6-methylated on one
416 16.7 Transcription
FIGURE 16.16 Thymidine dimer formation. Ultraviolet light catalyzes the joining of neighbor-
ing (vicinyl) thymidine bases, requiring a DNA repair process.
strand; the protein complex contains an endonuclease, which catalyzes the hydrolysis of the
daughter strand within the loop. Following removal of the bases, including the mismatch,
DNA polymerase I adds new bases, and the action of DNA ligase joins the remaining gap
in the phosphodiester backbone. A similar set of binding proteins and enzymes exists for
eukaryotic mismatch repair, although the process is less well understood. Nonetheless, it has
been established that one form of colorectal cancer in humans results from inherited muta-
tions in genes that code for proteins involved in mismatch repair.
16.6.2 EXCISION REPAIR
Excision repair corrects damage to existing DNA. For example, UV light catalyzes the for-
mation of thymidine dimers when these bases are adjacent in DNA sequences (Figure 16.16).
In this case, endonucleases – induced by ultraviolet radiation– catalyze the excision of the
surrounding DNA segments. If not repaired, such damage can cause skin cancers.
Other types of DNA damage requiring excision repair, such as radiation or pollutants,
involve distinctive repair systems. These repair systems also ensure DNA integrity during
transcription to form RNA.
16.6.3 PARP
An established form of DNA repair involves the enzyme Poly ADP-ribose polymerase, or
PARP. The mechanism of the best-studied isoform of this enzyme, PARP1 (here, just PARP)
is illustrated as Figure 16.17. DNA strand breaks are binding sites for the protein PARP, which
forms a dimer and catalyzes the addition of a portion of the coenzyme NAD+ to PARP itself.
This self-modification is reminiscent of the self-phosphorylation of the insulin receptor of
Chapter 13.
PARP catalyzes the attachment of the first NAD+ residue onto an aspartate side chain in
the illustration; other side chains (glutamate, serine, cysteine, or lysine) can also be used. The
reaction displaces the N-glycosidic bond of NAD+, releasing nicotinamide to form the link.
Further linkages are indicated using the ribose hydroxyl groups as nucleophiles; the chain
can grow to 30 residues and include branches.
The attached poly-ADP-ribose (ADPr) serves as a binding site for proteins involved in
DNA repair, for example, DNA polymerase III. Thus, PARP serves to both localize the DNA
damage and construct a binding site to recruit further repair proteins. PARP also serves to
signal other events in the cell, for example, control of the cell cycle and apoptosis (regulated
cell death).
A separate family of enzymes transfers just one ADPr unit on proteins, including target-
ing proteins for localization in the nucleus, into the ER, and for selective proteolysis by ubiq-
uitin (see Chapter 17). The most well-known cases of ADPr are those catalyzed by bacterial
toxins (Box 16.1: Cholera and Diphtheria).
16.7 TRANSCRIPTION
Transcription shares many features with replication. For example, producing RNA from
DNA requires opening the DNA duplex, a single-stranded DNA template, and polymerase
FIGURE 16.17 Mechanism of PARP. The PARP dimer binds damaged DNA; one of the acceptor sites (a carboxylate group from
asp) is shown. After binding DNA, the ADPr portion of NAD+ is transferred to one of the PARP dimers. The process is continued; a
second transfer is shown. The nicotinamide product of each transfer is illustrated in red.
Box 16.1 Cholera and Diphtheria Toxins
Two widespread diseases caused by bacterial toxins are cholera and diphtheria, which
cause selective ADPr modifications of eukaryotic proteins. Cholera toxin leads to cellular
uptake of an enzyme that inserts an ADPr (from NAD+) into the Gs-protein. This, in turn,
activates adenylate cyclase irreversibly, stimulating a chloride channel called CFTR (cystic
fibrosis transmembrane regulator). CFTR is named for the genetic deficiency disorder in
which secretions of the lung and pancreas are impaired. Cholera toxin targets mainly the
intestinal cells, leading to oversecretion of chloride and fluids and subsequent dehydra-
tion. Diphtheria toxin leads to cellular incorporation of a distinct ADPr modification, also
using NAD+ as the substrate for protein adenylation. In this case, the target is a step in
protein synthesis, an elongation factor (Chapter 17). Inhibition of the process leads to
cell death; susceptible targets include cardiac and neural cells. Cholera and diphtheria
toxins are not only the classic examples of NAD+ incorporation into ADPr, but they also
define the two distinct categories of ADP ribosylation mechanisms
activities progressing in a 5' to 3' direction. We have already been introduced to one RNA
polymerase, primase, which produces the RNA primer needed to begin DNA synthesis. RNA
polymerases are distinctive because they do not require an existing polymer, and only one
strand of DNA is copied. In transcription, only small portions of the genome are engaged at
any given time, and they are copied into RNA at different rates. Moreover, RNA is far less
stable than DNA, both chemically and biologically.
Several distinct types of RNA are formed in transcription (Figure 16.18). The expression of
cellular function is represented by messenger RNA (mRNA), which serves as the template
for protein synthesis by the process of translation. The scaffolding for this process – the
FIGURE 16.18 RNA families. The mRNAs are a branch by themselves because they code for
proteins. The noncoding RNAs involved in transcription are ribosomal (rRNA) and transfer (tRNA).
The small RNAs are regulatory: the small interfering RNAs (siRNA), the micro RNAs (miRNA), the
small nucleolar RNAs (snoRNA), and the small nuclear RNAs (snRNA).
FIGURE 16.19 Transcription initiation in E. coli. The first nucleotide copied into RNA is labeled
+1; the RNA polymerase binds upstream of that site, as indicated. Two upstream regions are indi-
cated that are widely found in prokaryotes, at −35 and at about −10 from the start site, which are
involved in binding and positioning RNA polymerase.
ribosome – contains ribosomal RNA (rRNA). Carrying amino acids and sequence recogni-
tion are the functions of transfer RNA (tRNA). A fourth class of small RNAs, such as the
siRNA, is involved in controlling cytosolic mRNA expression.
The structure of a segment of DNA in prokaryotes is illustrated in Figure 16.19. The direc-
tion of transcription is always from 5' to 3', but only one strand of DNA is a template for
creating the RNA transcript. When the RNA polymerase first binds to DNA, it recognizes
a site upstream of the first nucleotide in the RNA transcript. The sites in the DNA template
are labeled as illustrated in Figure 16.19: nucleotide #1 is the first to be copied into RNA,
and additional nucleotides in the RNA transcript are numbered sequentially. This positive
direction is considered downstream. Those nucleotides prior to the start site are labeled
sequentially as −1, −2, etc., and are upstream. Binding recognition results from distinctions
in the upstream portion of the DNA duplex structure that is known to be associated with
specific sequences, called promoters. Typically, these regions are specific gene sequences.
One that frequently occurs in bacteria is the TATAAT region (called a TATA box in eukary-
otes), which serves as a binding site for the RNA polymerase.
16.7.1 RNA POLYMERASE BINDING TO DNA
RNA polymerase binds selectively to one strand of DNA. In addition to RNA polymerase,
many other proteins recognize specific single-stranded DNA sequences, often six to eight
nucleotides long. This event may assist or hinder the binding of RNA polymerase itself.
While a specific sequence dictates the binding interactions of RNA polymerase, the initial
binding of the enzyme to DNA is nonselective, and RNA polymerase both dissociates and
scans along the surface until a selective binding site is encountered, locking the enzyme onto
the DNA.
16.7.2 TRANSCRIPTION EVENTS IN E. COLI
The basic events in the transcription of RNA – initiation, elongation, and termination – are
well studied in E. coli. RNA polymerase (RNA Pol) binds upstream of the start site. In the DNA
sequence of bacterial genes, many similar sequences (called consensus sequences) exist in two
upstream regions. One of these is the −35 region, and the other is the −10 region (also called the
Pribnow box, after its discoverer). These are the points of contact of RNA Pol and define the
promoters. Thus, RNA Pol binds DNA with relatively high affinity and close to the start site.
The initiating event requires dissociation of the holoenzyme RNA Pol from DNA. The
subunit structure of the holoenzyme is α2ββ’σ, which binds relatively weakly to the DNA
duplex and slides on its surface until it recognizes promoter elements. At this point, RNA
Pol binds tightly to the promoter elements, opens the DNA double helix (assisted by the fact
that the consensus sequence promoters have a high AT content), and synthesizes an RNA
transcript segment of about 10 nucleotides.
Next, the σ subunit dissociates, leaving the core complex α2ββ’, which binds less tightly to
DNA, and elongation ensues. The dissociated σ subunit can bind to another core and form a
separate holoenzyme to initiate another RNA transcript. As RNA polymerase proceeds, the
unwound DNA creates superhelix segments around it, resolved by topoisomerases.
Termination results from the formation of a hairpin loop in the RNA transcript itself, as
well as a termination sequence, as illustrated in (Figure 16.20). Hairpin loops form from
complementary base pairing in which a forward sequence is repeated in reverse; this is
known as a palindrome (Box 16.2). At the termination sequence, the RNA Pol dissociates,
and the transcript is complete. Alternatively, some RNA sequences require the participation
of separate proteins, most commonly the rho protein, which binds and disassembles the
RNA transcript with concomitant ATPase activity.
Transcripts of RNA in E. coli produce multiple components. For rRNA and tRNA forma-
tion, a single transcript can produce several of these components in a single chain that are
subsequently hydrolyzed (Figure 16.21). For mRNA formation, it is common to have several
genes coded in a single transcript, which have related functional activity. The entire tran-
script, called an operon, thus represents the precursor to a set of proteins that are formed
simultaneously. Figure 16.22 outlines the well-studied lac operon, which codes for a set of
enzymes involved in bacterial uptake and metabolism of lactose.
16.7.3 EUKARYOTIC TRANSCRIPTION
While the fundamental process of transcription is the same in both prokaryotes and eukary-
otes, several differences exist because eukaryotes divide events between organelles and
among different cell types. One reflection of this is the existence of three RNA Pol enzymes
(I, II, and III) in eukaryotes, which catalyze ribosomal (I), messenger (II), and transfer and
small (III) RNA transcription. Of the three, RNA Pol I and III are most similar to the prokary-
otic enzyme, in that both make a product that is subsequently cleaved into several smaller
individual RNAs. For example, RNA Pol I catalyzes the transcription of a pre-rRNA, which
is post-transcriptionally modified, and subsequently cleaved into 18S, 5.85S, and 28S rRNA.
These rRNAs then assemble with several proteins to form ribosomes, the scaffold assembly
for the synthesis of proteins. RNA Pol III catalyzes the synthesis of a precursor transcript
that folds into loops that, upon hydrolysis, forms tRNA and is also a precursor for other small
RNA transcripts, such as siRNA.
The most divergent form of eukaryotic transcription is catalyzed by RNA Pol II, which
catalyzes mRNA synthesis. Formation of the initiation complex by RNA Pol II is commonly
FIGURE 16.20 Hairpin formation effects transcription termination. As RNA polymerase pro-
ceeds, the enzyme is displaced as a hairpin loop, formed as a result of a palindromic sequence.
Box 16.2 Word Origins: Palindrome
A palindrome is a sequence of letters that reads the same in the forward direction as the
reverse. In the case of the RNA transcripts we have encountered, there are just four let-
ters (G, C, A, and U), and the reverse sequence can meet the forward sequence in three
dimensions, forming a double-stranded segment. This segment often forms a loop with
a sharp turn, called a hairpin loop. These loops are protein binding sites important in
regulation.
The word itself is of Greek origin, combining two words: palin (again) and drom (run). In
the English language, one example is “A man, a plan a canal – Panama”. A related usage is
in music, where the theme is run forward and then resolves itself in reverse. An example
is Haydn’s Palindrome Symphony (No. 47).
the rate-limiting step for overall protein synthesis, although other important levels of control
exist. Compared to prokaryotes, a greater number of proteins are involved in the complex,
and coregulators are common. For example, retinoids (Figure 16.23) are ligands for proteins
that bind DNA and regulate transcription. Another important level of control of DNA tran-
scription is the acetylation of histones, the proteins that wrap DNA. Acetylated histones
facilitate the opening of DNA and enable its transcription. Deacetylation of histones leads to
their re-association with DNA, inactivating transcription.
FIGURE 16.21 Formation of rRNA and tRNA from a single transcript in E. coli.
FIGURE 16.22 The lac operon. Three enzymes – galactosidase, permease, and transacetylase –
are required for the metabolism of galactose. mRNA formation for all of them is suppressed by
allolactose, metabolically derived from galactose. (a) Allolactose binds a protein repressor. The
complex can then bind to the operator region and prevent transcription of the genes for lactose
catabolism. (b) Reduction in lactose causes a reduction in allolactose, leading to dissociation
of the inducer-repressor complex, permitting RNA polymerase binding. Subsequently, the cell
forms mRNA for all three proteins.
Several processing reactions are unique to eukaryotic DNA formation. For example,
shortly after initiation, the first base is modified to form a cap structure. A molecule of
GTP is inserted as the first nucleotide in a 5’ to 5’ direction, as illustrated in (Figure 16.24).
A further modification of the original first nucleotide is methylation, originating from SAM.
At the 3' end of mRNA, the enzyme Poly A polymerase catalyzes the addition of as many
FIGURE 16.23 Retinoids. Common forms of retinoids in eukaryotes are retinal, the ester
retinyl palmitate, and retinal. The ester is cleaved in cells to form the active structure, the free
acid. The various oxidation states of retinoids are all regulators of cellular transcription, often as
co-activators.
FIGURE 16.24 The RNA cap. The 5' end of eukaryotic mRNA has a structure called a cap,
utilizing the cofactor S-adenosylmethionine and accounting for the methylated adenine at the
terminus.
as 250 adenine residues, forming the poly-A tail of the transcript. The final processing step
in the nucleus is removing intermediate portions of the transcript by splicing and rejoin-
ing to create the final mRNA. In some cases, different final products result from alternative
splicing. The segments removed are known as introns, whereas the pieces “spliced” together
to form the final transcript are known as exons (Figure 16.25). Recently, techniques have
been developed that attempt to directly sequence just the exons of genes (Box 16.3). Finally,
the finished mRNA exits through the nuclear pore to the cytosol, where translation occurs.
Once in the cytosol, both cap and tail structures help orient mRNA for protein translation
and protect the overall molecule from the action of cytosolic ribonuclease (RNase), which
catalyzes the degradation of RNA into smaller pieces. With the production of RNA, the cell
is poised to produce proteins, the subject of Chapter 17.
FIGURE 16.25 Exons and introns. The mRNA of eukaryotes contains both translatable RNA
(exons) and non-translatable RNA (introns). The introns are excised, and the exons are joined
together in the nucleus prior to exiting as a mature RNA into the cytosol.
Box 16.3 Exome Sequencing
There are about 20,000 genes in the human genome, but this number represents only 1%
of the total DNA. While the other segments have some known biological function, such
as promoter and enhancer regions, it would be advantageous to study just the small frac-
tion involved in protein synthesis.
Exome sequencing is a technique for separating the exons from the remainder of
the DNA and then sequencing just those nucleotides. The name is a nod to the origi-
nal systematic study of genetics (genomics), followed by the same name applied to pro-
teins (proteomics) and then to metabolism (metabolomics). As DNA is expressed in RNA,
the RNA is first isolated and then converted to a complementary DNA (cDNA) library.
Subsequently, cellular DNA is subjected to partial enzymatic hydrolysis of its phos-
phate esters and hybridized with cDNA (DNA copied in vitro from total cellular mRNA).
Sequencing of just the variously sized hybrids comprises the exome, which, depending
on the variant of the method used, represents the bulk of the exons.
Several rare diseases have already been related to their genes based solely on this
technique, such as identifying a defective gene for a chloride transporter in a kidney dis-
order. Another example is a loss of dihydroorotate dehydrogenase that was discovered
as the basis of Miller syndrome, a developmental disorder leading to abnormal facial and
limb features. It is significant that the enzyme is part of the pyrimidine biosynthesis path-
way. Known disorders in this pathway are distinct from Miller syndrome. Thus, exome
sequencing can reveal the underlying defect of multiple disorders.
SUMMARY
The nucleic acids DNA and RNA direct cellular replication, transcription, and translation.
DNA is a double-stranded polymer of nucleotides, linked in phosphate esters at the 3' and 5'
positions of the deoxyribose. The bases present are adenine (A), thymine (T), cytidine (C), and
guanine (G). The complete structure forms a “ladder” in which one side (chain) is arranged 5'
to 3', the other 3' to 5', and the “rungs” are a pair of bases hydrogen bonded together, either an
AT pair or a GC pair. RNA exists as a single polymer, with the same bases except that uracil
(U) is present in place of thymine. The double helix is stabilized by stacking interactions.
The base pairs above and below are attracted as partial positive charges from the edges of
the ring structures attract partial negative charges of π–clouds of the aromatic rings beneath
it. DNA is normally maintained in a supercoiled form, requiring topoisomerases to unravel
the molecule for replication or transcription. In addition, DNA is stored wrapped around
histones, basic proteins attracted to the negatively charged phosphate groups in the chain
backbone. For replication, the DNA opens into two separate chains and allows hybridization
of new nucleotides, in keeping with the AT and GC pairing rules. Each acts as a template,
so two daughter strands are formed. The process in bacteria originates at an origin that is
AT-rich; these base pairs are bound together less tightly than GC pairs. Proteins are required
to initiate and continue the process of replication, always in the direction of 5' to 3'. One of
the chains is synthesized linearly (leading strand), but the other (lagging strand) is formed in
small subunits because a new template only becomes available upstream of the direction of
replication. The actively replicating chain is held in a replisome, in which one of the strands
is looped. The structure enforces the same rate of synthesis of leading and lagging strands.
In eukaryotes, the process involves more proteins but is qualitatively different because these
DNA molecules are linear rather than circular. Sequences at the ends of the linear structures
are called telomeres, and require capping reactions to prevent shortening due to uneven
overhang during replication of the DNA. Errors incurred during DNA synthesis are resolved
by mismatch repair. Repair proteins bind the bulge at the mismatch site and thread the DNA
strand into a growing loop until a methylation site is encountered. A portion of the errant
daughter strand – recognized as new by being non-methylated – is digested by an endonucle-
ase within the protein complex. Several processes that degrade DNA, such as oxidation or
UV damage, can be restored by excision repair. Segments are removed by hydrolysis of the
phosphate backbones and filled in by polymerase and ligase. One type of excision repair is
PARP, which catalyzes a chain of ADP-ribose units as a recognition site for binding repair
proteins.
The transcription process forms RNA from a DNA template. One form of RNA ultimately
codes for protein: mRNA. rRNA forms part of the protein synthesis scaffold in the cytosol
(the ribosome), tRNA ferries amino acids to the translational complex and the other small
RNA species serves to regulate transcription and translation. Transcription requires bind-
ing of RNA polymerase to a nucleotide sequence that is upstream of the start site and pro-
ceeds in a 5' to 3' direction. Regulation of transcription in bacteria occurs at the formation of
the initiation complex. The initiation complex is also the point of regulation in eukaryotes,
although the control process is more involved, with multiple components needed to regulate
transcription for individual mRNA transcripts. A further distinction of eukaryotes is the
presence of introns within a transcribed mRNA; these sequences must be excised and the
remaining pieces of mRNA – the exons – spliced together. Prokaryotic mRNA is polycis-
tronic so that several proteins can be formed from a single pass by polymerase. Eukaryotic
mRNA is monocistronic, and the mRNA is modified by methylation in the first nucleotide,
the cap, and addition of multiple adenosyl residues to the 3' end, the poly-A tail.
REVIEW QUESTIONS
1. Why do prokaryotes generally have no systems for direct control of protein synthesis?
2. One way antibody diversity is achieved is through the enzyme cytidine deaminase,
which removes the ammonia from a cytidine residue. After deamination, what would
be the complementary base?
3. Okasaki fragments must be joined by a ligase reaction. What would be the enzyme
intermediate for this reaction?
4. Why are AT base pairs such as those present at the replication origins intrinsically
weaker than GC base pairs?
5. The lac operon, a model for transcription regulation, produces a single mRNA that
codes for three proteins. Does this mean the rate of formation of all three proteins
must be the same?
6. What is the structural basis for the major and minor grooves in DNA?
7. Most of the time, DNA exists as a double strand. Enumerate the circumstances under
which it is a single strand.
8. The enzyme PPAR is a common target for anticancer drugs. Why is this an appropri-
ate therapy?
9. What is the purpose of palindromic sequences?
10. Under what conditions would base stacking interactions be significant in RNA?
CHAPTER 16 ADDENDUM: RESTRICTION ENZYMES AND PCR
Of the many tools of molecular biology, two are pivotal to the expansion of this field: restric-
tion enzymes and the polymerase chain reaction or PCR. Here we will examine the origin
of these methods and their utility.
RESTRICTION ENZYMES
The unusual name for this class of enzymes comes from the biological description of the
bacterial defense system against bacteriophages (or just phages). The phages are viruses that
specifically infect bacteria. To counter phage invasion, bacteria encode enzymes that cata-
lyze viral DNA hydrolysis, thereby restricting the range of prey for the virus.
Restriction enzymes bind specific sequences, usually of a few nucleotides long, such as
6 or 8, and subsequently cleave the DNA. Bacteria also synthesize a DNA methylase of the
same specificity to protect their own genome against digestion from the restriction enzyme.
The purified restriction enzymes available today target hundreds of specific sequences. The
enzymes enable investigators to “cut” DNA selectively, introduce a distinct sequence into a
known site, and “paste” it together using DNA ligase. Restriction enzymes are used in vir-
tually every phase of research on nucleic acids. Other discoveries resulting from phage–
bacteria interactions have more recently been made. One of them is the cloning technique
abbreviated as CRISPR (clustered regularly interspaced short palindromic repeats). While
this elaborate bacterial defense system also leads to cleavage of foreign DNA, it is more anal-
ogous to higher organisms’ immune defense.
PCR
The discovery of PCR in the early 1980s led to a renaissance in molecular biology, as it dras-
tically reduced the time required to produce prodigious amounts of DNA from very small
amounts of starting material. Beyond this, its wide use in forensics and medical diagnosis has
turned a formerly breakthrough technique into a routine analysis.
The background to the method is the copying of DNA molecules in vitro, once a tedious
task of melting the DNA double strands, including a primer that has a base pair comple-
mentary to a sequence of interest and then synthesizing a larger strand, using the original
opened piece as a template. The problem encountered was that the polymerase, being a pro-
tein, was destroyed by the heating process necessary to break the hydrogen bonds of the
double stranded DNA, making the repetition of the procedure difficult.
This issue was resolved by using a distinct polymerase isolated from thermophiles,
organisms from certain archaea or bacteria that thrive at very high temperatures. These
could survive the very high temperatures needed to open the strands. Thus, cycles of heating
and cooling – with the latter having the attended growing chains – could produce exponen-
tially increasing amounts of new DNA strands. For example, in the analysis of the COVID-19
virus, accurate determination of the presence of nucleic acid fragments specific to the virus
even at vanishingly small concentrations is possible with the PCR technique.
From a biochemical standpoint, the curious issue is how the thermophiles survive such
high temperatures. There are several modifications of protein, nucleic acids, and lipids. For
example, proteins of thermophiles have a more compact structure, more extensive hydrogen
bonding, and greater proportions of hydrophobic cores. Nucleic acids have a greater GC
426 Key Terms
content and correspondingly higher DNA melting points. Ether lipids are more prominent,
as these are chemically more stable.
Thermophiles are just one example of organisms that thrive in extreme environments; the
general term is extremophiles. Thus, some organisms survive very cold temperatures (less
than 15o C), extremes of pH (such as the tilapia of Chapter Appendix 15), high salinity, and
high pressures.
KEY TERMS
cap
capping
complementarity
consensus sequences
core complex
CRISPR
DNA gyrase
DNA ligase
DNA polymerase
double helix
downstream
elongation
excision repair
exome sequencing
exons
exonuclease
extremophiles
5' end
hairpin
hemimethylated
histone
introns
lac operon
lagging strand
leading strand
loop
major groove
melting temperature
minor groove
messenger RNA
mismatch repair
Okazaki fragments
palindrome
poly ADP-ribose polymerase (PARP)
poly A polymerase
poly-A tail
polyamines
polymerase chain reaction
Pribnow box
primase
primosome
processivity
promoters
replication
replication fork
replisome
restriction enzymes
rho protein
ribosomal RNA
single-stranded binding protein
siRNA
stacking interaction
supercoils
TATA
telomeres
termination sequence
thermophiles
topoisomerases
transcription
transfer RNA
upstream
BIBLIOGRAPHY
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M.S. Cohen, P. Chang. Insights into the Biogenesis, Function, and Regulation of Adp-Ribosylation.
Nature Chemical Biology 14 (2018) 236–243.
J.A. Coker. Recent Advances in Understanding Extremophiles. F1000Res 8 https://doi.org/10.12688/
f1000research.20765.1 (2019).
L. Garibyan, N. Avashia. Polymerase Chain Reaction. Journal of Investigative Dermatology 133 (2013)
1–4.
M. Jinek, K. Chylinski, I. Fonfara, M. Hauer, J.A. Doudna, E. Charpentier. A Programmable Dual-
Rna–Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science 337 (2012) 816–821.
J. Kaiser. Human Genetics. Affordable 'Exomes' Fill Gaps in a Catalog of Rare Diseases. Science 330
(2010) 903.
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Art Genetic Manipulation Tool. Antibiotics 8 (2019) 18.
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Mendelian Disorder. Nature Genetics 42 (2010) 30–35.
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Biotechniques 69 (2020) 317–325.
Protein Synthesis
and Degradation 17
Our final topics are protein synthesis and protein degradation. Bacterial protein synthe-
sis can be divided between initiation, elongation, and termination. While not fundamen-
tally different, the eukaryotic process involves more regulatory proteins, a larger assembly
complex (ribosome), and more elaborate regulation mechanisms. Bacteria synthesize pro-
teins even before the mRNA is fully synthesized and produce several proteins from a single
mRNA. Hence, protein translation is not regulated separately from transcription in these
organisms. The primary process for cellular protein degradation of eukaryotes involves a
molecular complex called the proteasome. Proteins are targeted for degradation at widely
different rates. Just like small-molecule intermediates, proteins themselves exist in a steady-
state between synthesis and degradation. The rate of protein degradation largely determines
protein accumulation. We begin our study of protein formation by examining the classes of
molecules involved and the genetic code underlying protein translation.
17.1 THREE FORMS OF RNA EMPLOYED IN PROTEIN SYNTHESIS
Transfer (tRNA), ribosomal (rRNA), and messenger (mRNA) RNA are transcribed from a
DNA template. In eukaryotes, these forms of RNA are exclusively synthesized in the nucleus
and exit through nuclear pores into the cytoplasm. These three forms of RNA all interact
during protein synthesis. We first consider the RNAs individually.
17.1.1
tRNA
Transfer RNA is relatively small, having about 80 nucleotides in the mature molecule. Three
views of a tRNA molecule are shown in Figure 17.1. The planar view of Figure 17.1a shows
extensive complementary base pairing, with three hairpin loops in a cloverleaf pattern.
Several of the bases are modified, most commonly by methylation. Figure 17.1b is a three-
dimensional view of tRNA. The twisting apparent in Figure 17.1b results from the same
stacking interactions that cause helix formation in DNA (Chapter 16). Two key regions of the
molecule are identified in each view: a three-base region called the anticodon site, and the
3' end, which terminates in CCA. The last ribose of the CCA end has a 5' hydroxyl group that
is esterified to the carboxyl group of an amino acid. This portion is called the acceptor stem.
The abbreviated view in Figure 17.1c shows only the anticodon region and the acceptor stem.
The attachment of a tRNA to an amino acid is a reaction catalyzed by one of over 20 spe-
cific amino-acyl-tRNA synthetases. The generic reaction is:

(17.1) Amino acid + tRNA + ATP ® tRNA amino acid + AMP + PPi
Specificity in catalysis for one of the synthetases involves several regions of the tRNA mol-
ecule; in some cases, even the anticodon loop binds the enzyme. The reaction mechanism,
shown in Figure 17.2, is similar to that of asparagine synthetase (Chapter 8). The enzyme first
binds an amino acid and ATP, forming a carboxyphosphate adduct with the α-phosphate
of ATP, releasing pyrophosphate. Subsequently, the CCA–OH group of the tRNA displaces
429
430 17.1 Three Forms of RNA Employed in Protein Synthesis
FIGURE 17.1 Three views of tRNA. (a) The two-dimensional representation shows extensive
intrachain hydrogen bonding. (b) Twisting in three dimensions is the result of stacking interac-
tions. (c) A symbolic view shows the anticodon and acceptor sites.
FIGURE 17.2 Mechanism of tRNA synthetase. In the first step, an acid phosphate forms between the carboxyl group of the
amino acid and the γ–phosphate of ATP. In the second step, the acceptor site of tRNA forms an ester with the amino acid, displac-
ing AMP.
Chapter 17 – Protein Synthesis and Degradation 431
AMP, attaching the amino acid. A tRNA with an amino acid attached is written as
tRNAamino acid and is called a charged tRNA. This conjugate joins the complex responsible for
protein synthesis.
Aside from the 20 amino acids, an additional tRNA attaches to methionine. This molecule
initiates all protein synthesis and is distinct from the tRNA Met species used for incorporat-
ing Met into the interior portions of proteins. In bacteria, this special tRNA Met is formylated,
using 10-formyl-THF as the precursor, to produce f-Met-tRNA.
17.1.2
rRNA AND THE RIBOSOMES
A planar view of the rRNA structure is shown in Figure 17.3. Like tRNA, extensive base pair-
ing in rRNA produces a more elaborate structure than the linear sequence would indicate.
A complex of proteins and rRNA comprises the ribosome, the scaffold upon which proteins
are synthesized. As shown in Figure 17.4, the ribosomes can dissociate into two complexes.
The prokaryotic ribosome is a 70S complex that decomposes into 30S and 5OS complexes,
FIGURE 17.3 Planar view of rRNA. Like tRNA, the molecule has extensive intramolecular base
pair bonding.
FIGURE 17.4 Bacterial and eukaryotic ribosomes.

432 17.2 The Genetic Code
Box 17.1 Unit Origins: The Svedberg
A few molecular complexes, such as ribosomes and proteasomes, are measured in sved-
berg units, abbreviated S. This is not an SI unit agreed upon by chemical commissions but
rather a curious holdout from a technique used to measure mixtures of macromolecules,
historically called colloids. The unit itself honors Theodor Svedberg (as with other epon-
ymous units, such as the newton and the joule). Svedberg invented the ultracentrifuge,
the device used to measure sedimentation coefficients for colloids like ribosomes. The
svedberg is a time unit: it is the ratio of velocity to acceleration in a centrifugal field. This
unit is useful because the particle achieves terminal velocity when the centrifugal force
is balanced by the viscosity of the media, just as the terminal velocity is achieved by a
parachutist when the acceleration due to gravity matches the wind resistance. The value
of 1 S is 10 −13 seconds. It measures particles, although it is not just size, but also shape-
dependent. Thus, the svedberg units are not additive, explaining why the bacterial 30S
and 50S particles combine to make a 70S ribosome.
whereas the eukaryotic 30S ribosome dissociates into 40S and 60S complexes. The unit of
measure for these complexes is the svedberg, based on the mobility of particles during ultra-
centrifugation (Box 17.1).
Eukaryotic ribosomes are a more extensive complex of rRNA and protein. In both pro-
karyotes and eukaryotes, the quantity of RNA in the ribosome exceeds that of protein. The
RNA is also used as a catalyst for the actual joining reaction that forms the peptide bond.
Ribosomes that act as enzymes are called ribozymes, an exception to the rule that enzymes
are proteins.
The ribosome is the site of convergence of rRNA, mRNA, and tRNA. The three bases of
the anticodon region in tRNA are base-complementary to the codon, a triplet of bases in
the mRNA bound to the ribosome. Each sequentially recognized codon corresponds to a
specific amino acid. The matching of codons to their amino acids is known as the genetic
code.
17.2 THE GENETIC CODE
The genetic code was deciphered through experiments in which protein synthesis was rep-
licated in vitro. The essential finding was that a mixture of ribosomes, tRNA molecules
attached to amino acids, and mRNA could assemble and form a protein chain. The key to
the translational machinery is the tRNA molecule. The anticodon of tRNA binds the codon
– a complementary triplet of bases in mRNA – and the amino acid on the opposing accep-
tor stem of tRNA forms a new peptide bond. Repeated many times, this forms the protein
chain. The specific amino acid incorporated is determined by the anticodon of the tRNA.
Early experiments used synthetic mRNA as the input and analyzed the amino acid sequence
of the resulting protein. For example, a chain composed only of adenosine bases (a poly–A
mRNA) produced polylysine, showing that AAA corresponds to Lys. More elaborate tech-
niques along the same lines led to the elucidation of the entire three-base sequence code,
shown in Table 17.1, which is nearly universal across species. The total number of possible
amino acids that could be coded by using three different nucleotides, where each may be one
of four possibilities (A, G, C, or U), is 4 × 4 × 4 = 64. Only 20 unique codons are necessary
for amino acids, as well as a few more triplets to code for termination – the termination
codons – so the genetic code is redundant. Variation in the third position of the anticodon
often results in the insertion of the same (that is, the correct) amino acid, which should be
apparent from Table 17.1 (note, for example, that CGX, where X = U, A, C, or G, codes for
Arg). This slight lapse of complete specificity – that the first two bases are more important
for complementary base recognition – is known as the wobble hypothesis.
TABLE 17.1 The Genetic Code

U C A G
U UUU Phe UCA Ser UAU Tyr UGU Cys U
UUC Phe UCC Ser UAC Tyr UGC Cys C
UUA Leu UCA Ser UAA STOP UGA STOP A
UUG Leu UCG Ser UAG STOP UGG Trp G
C CUU Leu CCU Pro CAU His CGU Arg U
CUC Leu CCC Pro CAC His CGC Arg C
CUA Leu CCA Pro CAA Gin CGA Arg A
CUG Leu CCG Pro CAG Gin CGG Arg G
A AUU ile ACU Thr AAU Asn AGU Ser U
AUC ile ACC Thr AAC Asn AGC Ser C
AUA ile ACA Thr AAA Lys AGA Arg A
AUG Met or START ACG Thr AAG Lys AGG Arg G
G GUU Val GCU Ala GAU Asp GGU Gly U
GUC Val GCC Ala GAC Asp GGC Gly C
GUA Val GCA Ala GAA Glu GGA Gly A
GUG Val GCG Ala GAG Glu GGG Gly G
a The Codon Triplet is shown starting at the 5′ end. The first nucleotide is
in the left hand column, the second in the top row, and the third along
the right-hand column.
17.3 STEPS IN PROTEIN SYNTHESIS
The overall process of protein translation is commonly divided into initiation, elongation,
and termination steps. Each requires a distinct set of complexes and regulatory features. We
focus first on bacterial protein synthesis, after which we consider key distinctions that exist
for eukaryotes.
17.3.1 INITIATION
In bacteria, initiation begins with mRNA binding to the smaller, 30S ribosomal subunit
(Figure 17.5). mRNA contains a 4-6 nucleotide sequence upstream of the start site called the
Shine–Dalgarno segment. rRNA within the 30S complex has a sequence complementary to
the Shine–Dalgarno segment, positioning the mRNA onto the rRNA. Two binding proteins –
IF-1(initiation factor-1) and IF-3 – attach to form the complex illustrated in Figure 17.5a. IF-1
prevents the binding of any tRNA species apart from the initiator, tRNA f-met. IF-3 prevents
the premature association of the 30S with the 50S complex. The binding of tRNA f-met is facili-
tated by the presence of the G-protein IF-2, in its active, GTP-bound form (Figure 17.5b). The
resulting complex (Figure 17.5c) can now bind the 50S subunit, which acts as a GTPase for
IF-2, leading to the release of all initiation factors, and leaving the tRNA f-met bound to the
complete ribosomal assembly (Figure 17.5d).
The complex now has two sites within the full 70S ribosomal assembly: the P site (pepti-
dyl), and the A site (acceptor); the tRNA f-met is in the P site. The complete initiation complex
is ready to undergo elongation.
17.3.2 ELONGATION
The steps of elongation are illustrated in Figure 17.6. Starting with the completed initia-
tion complex, a charged tRNA binds to the adjacent codon, positioning the two amino acids
434 17.3 Steps in Protein Synthesis
FIGURE 17.5 Initiation of translation. (a) The 30S component binds initiation factors IF-1 and
IF-3 and mRNA to form a complex. IF-1 binds the A site, blocking incoming tRNA molecules. IF-3
prevents premature binding to the 50S subunit. (b) IF-2 (a G-protein) and f-Met-tRNA bind at
the initiation (AUG) site on the mRNA (c). The 50S subunit binds to this complex, catalyzing the
GTPase activity of IF-2. Subsequently, all of the initiation factors are released, and the complete
initiation complex forms (d).
together, filling the P and A sites (Figure 17.6a). Binding of the incoming charged tRNA to
the A site is assisted by EF-Tu, an elongation factor G-protein that undergoes GTP hydrolysis
upon binding, releasing the EF–Tu in its GDP-bound form. In the next step, the peptide bond
forms (Figure 17.6b), catalyzed by rRNA rather than a protein (Figure 17.7 shows the mecha-
nism). The amine from the amino acid at the A site displaces the esterified f-Met-tRNA at
the P site. Thus, the newly formed dipeptide becomes attached to the tRNA at the A site,
leaving an empty tRNA at the P site (Figure 17.6c). Next, binding of the elongation factor
FIGURE 17.6 Translation elongation. The initiated ribosome (a) binds the activated tRNA bear-
ing the second amino acid, assisted by EF-Tu in the GTP form (a). Once the A site is bound to this
activated tRNA (b), the EF-Tu:GDP (and Pi) is released. Next, the amine of aa2 displaces the f-met
from its acceptor site, forming a peptide bond (c). Finally, EF-Ts:GTP binds, shifting the mRNA over
by three nucleotides, releasing the now free tRNA, and leaving the tRNA containing the short
peptide in the P site (d). The cycle then resumes with the next incoming amino acyl-tRNA able
to occupy the A site.
EF-Ts promotes mRNA movement along the ribosome so that the tRNA appears at the P
site (Figure 17.6d). This action also displaces the first tRNA from the ribosome, leaving the A
site empty and a dipeptide attached to the tRNA in the P site. This complex enters into the
second round of elongation, and the cycle is repeated until a triplet codon is encountered for
which no tRNA exists; this is the termination codon.
While fMet-tRNA is used for the first amino acid, the internal Met residues of the protein
use a distinct tRNA. Hence, only the f-Met-tRNA can directly enter what becomes the P site,
and all other amino acids enter the A site. The rationale for using the formylated methionine
436 17.3 Steps in Protein Synthesis
FIGURE 17.7 Mechanism of amino acyl-tRNA synthase. The amine of the aminoacyl group in
the A site initiates a nucleophilic attack on the acyl group of the aminoacyl group in the P site,
leaving a free tRNA in the P site and a new peptide bound in the A site.
is to prevent the reaction of the amino end of the first amino acid with the ester of the sec-
ond. Subsequent methionine residues cannot be formylated because they would be unable to
elongate the protein chain.
17.3.3 TERMINATION
Upon encountering a termination codon, another protein (a release factor) binds the com-
plex and catalyzes the hydrolysis of the ester bond linking the completed protein. The ribo-
somal assembly then dissociates into the 30S and 50S subunits. Subsequently, the ribosome
complex reforms, and the entire cycle of translation is repeated at other sites. Because bac-
terial mRNA is polycistronic – having sequences for several proteins in the same mRNA
strand – this cycle can continue on a downstream segment of the same mRNA. Moreover, in
bacteria, the process of translation can begin well before transcription has ended.
17.3.4 DISTINCTIVE FEATURES OF EUKARYOTIC TRANSLATION
While translation is similar in eukaryotes and bacteria, more proteins are involved in
eukaryotes, particularly during initiation. Additionally, the synthesis of eukaryotic mRNA
(a nuclear event) takes place in a separate compartment from translation (a cytosolic event),
and eukaryotic mRNA is monocistronic (it codes for just one protein).
There are no Shine–Dalgarno sequences in eukaryotic mRNA. Instead, the initial posi-
tioning of the mRNA relies upon several eukaryotic initiation factors – eIF proteins – as
well as the 5'-cap structure and the 3'-poly-A tail of the mRNA. Thus, the initiation complex
consists of eIF proteins at the beginning and end segments of the mRNA. The protein assist-
ing the first incoming tRNA – which is a Met-tRNA – is also a G-protein, analogous to the
one in bacteria. In eukaryotes, it is eIF-2. Once bound, the proteins assist the tRNA in scan-
ning along the mRNA for the first AUG codon sequence, typically the initiation point for
translation. Like prokaryotes, hydrolysis of the bound GTP of eIF-2 leads to the dissociation
of the proteins on the small ribosomal unit and the formation of the full ribosome assembly.
Eukaryotic elongation factors – called eEF proteins – similarly assist the incorporation of
incoming charged tRNAs. The formation of the protein continues until a termination codon
is encountered, similar to the prokaryotic system.
17.3.5 REGULATION OF EUKARYOTIC TRANSLATION
Because mRNA is translated even as it is being synthesized in prokaryotes, there is little

opportunity for separate control of the translation process. This kind of regulation does
occur in eukaryotes, however.
One example is the control of hemoglobin protein synthesis in reticulocytes by heme or
iron. Reticulocytes have no internal compartments and no nucleus to produce new mRNA.
A decrease in heme or iron concentration leads to the activation of a protein called heme
regulated inhibitor (HRI). HRI is itself a protein kinase that catalyzes the phosphorylation
of eIF-2, which is a G-protein (Figure 17.8). The form that is a substrate of HRI is the inactive,
GDP-bound form. This species binds tightly to eIF2B, thereby sequestering eIF2B. Because
eIF2B is the catalyst that activates eIF2 by displacing GDP in favor of GTP, the overall result
of HRI is the inactivation of initiation, and hence protein synthesis. In this way, HRI dimin-
ishes hemoglobin synthesis under conditions of diminished heme or iron availability.
17.4 CO-TRANSLATIONAL AND POST-TRANSLATIONAL

MODIFICATIONS OF PROTEINS
17.4.1 CO-TRANSLATIONAL MODIFICATIONS
Co-translational modifications of proteins include the de-formylation of the leading f–Met

residue and the hydrolysis of one or more amino acids from the N terminus of the protein, an
activity catalyzed by aminopeptidase.
Protein folding, another co-translational modification, begins shortly after a stretch of
amino acids emerges from the ribosome. This process is assisted by a class of proteins called
chaperones (introduced in Chapter 5). Chaperones also catalyze the refolding of fully syn-
thesized proteins. One common consequence of structural misalignment arises from proline
residues that are nonenzymatically rearranged from the physiological trans configuration, in
which neighboring side chains extend in opposing spatial directions, to the cis configuration.
The flipping occurs because proline has a relatively low energy barrier for this nonenzymatic
transformation. The enzyme, cis-trans prolyl isomerase, lowers this barrier even further,
which allows the ultimately more stable trans configuration to prevail, thus favoring normal
protein folding (see the mechanism in Figure 17.9). Certain cyclic compounds, such as cyclo-
sporin, can bind the active site of prolyl isomerases, inhibiting their activity. Surprisingly, the
cyclosporine-prolyl isomerase complex itself serves as a selective inhibitor of an unrelated
protein that leads to immunosuppression (Box 17.2).
438 17.4 Co-Translational and Post-Translational Modifications of Proteins
FIGURE 17.8 Heme-regulated inhibitor (HRI) control of protein translation. In the normal case
(left panel), eIF2 becomes activated by the G-activating protein eIF2B. However, when either
heme or iron is in low concentration, HRI activity is elevated. HRI catalyzes the phosphorylation
of eIF2:GDP, which tightly binds and sequesters eIF2B.
FIGURE 17.9 Mechanisms of cis-trans prolyl isomerase. The enzyme provides a binding site
that stabilizes the flat intermediate between the cis and trans forms. This activity increases the
interconversion of the two forms. The trans form is more stable, so enzyme catalysis results in an
increase in the trans form.
Box 17.2 Macromolecular Inhibitory Complexes
An unusual type of inhibition is exerted by certain cyclic compounds (Figure B17.2) that
form a complex with the enzyme cis-trans prolyl isomerase. Tacrolimus and rapamycin
both have a flattened prolyl residue that tightly binds the active site of the prolyl isom-
erase. However, because the residue is part of a small cyclic peptide, the enzyme can-
not catalyze its reaction, thus forming a dead-end complex. Cyclosporin has no proline
residue, but its structure nonetheless fits the prolyl isomerase active site and binds the
enzyme tightly. The cyclosporin-prolyl isomerase complex inhibits the protein phospha-
tase calcineurin. Calcineurin catalyzes the dephosphorylation of a transcription factor
that prevents the formation of the T-cell-derived protein, interleukin-2 (IL-2). The absence
of IL-2 prevents further proliferation of helper T cells, leading to immunosuppression.
For this reason, the prolyl isomerases are sometimes called immunophilins. A second
compound, FK506, binds a distinct prolyl isomerase; the FK506- prolylisomerase complex
also inhibits calcineurin, with an order of magnitude greater potency than cyclosporin.
Rapamycin, on the other hand, binds prolyl isomerase and inhibits its namesake target,
mTOR. Rapamycin was discovered in a species of Streptomyces on an island in the south-
ern Pacific Ocean, 1500 miles west of Chile, known by outsiders as Easter Island but by its
indigenous people as Rapa Nui.
FIGURE B17.2 Antibiotics that bind prolyl isomerase.

440 17.5 Protein Degradation
17.4.2 POST-TRANSLATIONAL MODIFICATIONS
Once a protein is fully formed and released from the ribosomal assembly, it may still be
subject to further modifications. These alterations – post-translational modifications –
are covalent alterations to proteins. We can divide these transformations into two classes,
pathway-reversible and pathway-irreversible.
Protein phosphorylation is a common type of post-translational modification (for exam-
ple, pyruvate kinase in Chapter 9 and glycogen synthase in Chapter 13) and is pathway-
reversible. The phosphorylation itself is not reversible in a chemical equilibrium sense, but a
separate enzyme – a protein phosphatase – can revert these enzymes to their original forms.
Thus, the reversion can be accomplished by a distinct reaction without the requirement for
new protein synthesis. Similarly, histones can be acetylated and deacetylated (Chapter 16),
altering the availability of DNA for transcription.
An example of a pathway-irreversible modification is the incorporation of sugar residues
into proteins destined for export from the cell. These reactions occur in the endoplasmic
reticulum and Golgi apparatus, and they produce proteins that are more viscous and resist
degradation in the extracellular environment. Similarly, the hydroxylation of proline resi-
dues in collagen leads to a more tightly coiled structural protein. These incorporations are
permanent modifications of proteins, and not removed until the protein is degraded – that
is, by proteolysis.
In some cases, pathway-irreversible modifications can be so extreme as to involve degra-
dation of most of the protein to produce a small molecule product. For example, thyroxine
formation involves the joining of tyrosine residues of the protein thyroglobulin, followed by
iodination of the aromatic hydroxyls of these tyrosines. Proteolysis then releases the modi-
fied dipeptide thyroxin (tetraiododityrosine). Similarly, creatine (the carrier of high-energy
phosphates in nerve and muscle) and carnitine (the carrier of fatty acids into the mitochon-
dria) are both formed by modifying an aminoacyl group of a protein, followed by the release
of these molecules due to protein degradation. We next consider the process of protein deg-
radation more broadly.
17.5 PROTEIN DEGRADATION
As we have just seen, protein degradation may be viewed as a modification of proteins or a

source of specific small molecules, such as thyroid hormone or carnitine. In some cases, pro-
teins are only partially degraded. For example, the amino terminus of some proteins serves
as a signal sequence to guide them into specific organelles, such as the endoplasmic reticu-
lum or mitochondria. Once the protein has entered the new location, this portion is removed
by proteolysis. Degradation may occur even as a protein is being synthesized, a strategy that
eliminates misfolded proteins. However, proteins are usually fully degraded, and the amino
acids are recycled, which we consider next.
17.5.1 EXTRACELLULAR PROTEASES
The earliest discovered proteases were those found in extracellular environments. For exam-
ple, the proteolytic enzyme trypsin, found in the stomach, catalyzes the partial hydrolysis
of proteins. Once the stomach chyme enters the intestine, several proteases secreted from
the pancreas further digest the proteins and protein fragments to produce amino acids and
some dipeptides. The mechanisms of the proteases have been well studied. One major class
is the serine proteases. These employ a triad of amino acids to activate the serine hydroxyl
for nucleophilic attack. A similar triad is used in acetylcholine esterase (Chapter 6). The
triad used by serine proteases is illustrated in Figure 17.10, and the mechanism for protease-
mediated hydrolysis of the peptide bond is shown in Figure 17.11.
A related class of proteases uses cysteine (rather than serine) as a nucleophile; others use a
metal ion to activate the amide for attack by water. Some selectivity exists in these proteases,
FIGURE 17.10 The serine protease triad. (a) The triad of amino acid residues – asp, his, and
ser – is hydrogen-bonded within the enzyme active site. The serine -OH group can initiate the
nucleophilic attack simultaneously as hydrogen is abstracted by the his residue; this concerted
reaction prevents a buildup of negative charge that would occur if the reaction were sequential.
(b) Abbreviated structure of the essential his and ser residues involved in catalysis.
FIGURE 17.11 Mechanism of serine protease. The imidazole of a histidine residue assists the hydroxyl group of serine in the
nucleophilic attack and provides an electron sink for subsequent electron rearrangement that leads to the cleavage of the pep-
tide bond. Next, the imidazole assists in both the hydration and hydrolysis of the enzyme-bound carboxyl group of the remain-
ing protein.
442 17.5 Protein Degradation
directed by the functional groups near the amide that is to be split. For example, chymo-
trypsin is relatively selective for aromatic residues on the N side of the peptide bond. Using
the known specificities of the proteases, early protein-sequencing studies were able to map
peptides and eventually the amino acid sequence of proteins.
Another important class of extracellular proteases is involved in the clotting reaction
involving a series of enzymes that catalyze the sequential peptide bond hydrolysis and ulti-
mately create a protein plug that prevents the leakage of blood vessels. Because each step
catalyzes many copies of the product, the sequence is an amplification that rapidly produces
large amounts of clotting material. A common form of hemophilia results from the genetic
loss of one of these enzymes, called factor VIII.
17.5.2 INTRACELLULAR PROTEASES
Organelles are degraded and recycled by fusing them with the lysosome, which has an acidic
environment produced by a proton pump analogous to the F1Fo ATP synthase of mitochon-
dria and chloroplasts. A group of proteases with an optimal pH in the acidic range catalyze
the degradation of the protein component of these organelles. Self-degradation (autophagy)
and vesicular uptake are examples of cellular activities that involve lysosome fusion.
Proteolytic degradation of cytosolic proteins requires a separate particle, the proteasome
(Figure 17.12a). This structure is a complex of proteins that creates an interior environment
separated from the cytosol in which proteins can be degraded once they are threaded into
it. Unfolding depends upon a post-translational modification called ubiquitination, in which
molecules of ubiquitin (a small 76-amino-acid protein, Figure 17.12b) are covalently attached
to proteins. Proteins that unfold become substrates for attachment to ubiquitin (UbiQ) by
the reaction outlined in Equation 17.2:
(17.2) UbiQ + ATP + E-Lys-NH 2 ® E-Lys-N-C(O)-UbiQ + AMP + PPi
At least four copies of ubiquitin must be added to a protein to allow it to recognize, bind to,
and thread itself into the cavity of a proteasome.
While many different proteins are degraded by the proteasome, they do so at distinctive
rates. As a result, the degradation half-lives for different proteins vary over a wide range, as
shown in Table 17.2. The shortest known half-life is that of ornithine decarboxylase, which
catalyzes the formation of putrescine in the polyamine pathway (Figure 17.13). Polyamines
bind the negatively-charged phosphate residues of DNA (Chapter 16). An enzyme with a
relatively short half-life is hepatic PEP carboxykinase, which catalyzes the rate-limiting step
of gluconeogenesis; its half-life is a few hours. Other proteins have half-lives that can range
up to many days or weeks.
FIGURE 17.12 The proteasome and ubiquitin. (a) The core particle of the proteasome is the
cylindrical; the hydrolysis of proteins occurs in its interior. Protein complexes at both ends cap the
cylinder, serving as selectivity filters. (b) Ribbon view of ubiquitin; when a string of at least four of
these molecules attach covalently to a protein, it is targeted to and degraded by the proteasome.
TABLE 17.2 Protein Half-Livesa

Protein Tissue Half-Life
Ornithine decarboxylase Rat liver 11 min
RNA polymerase I Rat liver 1.3 h
Phosphoenolpyruvate carboxykinasec Rat liver 6h
Hexokinase Rat liver 1 day
Acety-CoA carboxylase Rat liver 2 days
Arginase Rat liver 5 days
Myosin (heavy chain) Rat heart 6 days
Myosin (light chain) Rat heart 9 days
Tropomyosin Rabbit skeletal muscle 25 days
Myosin Rabbit skeletal muscle 30 days
Actin Rabbit skeletal muscle >50 days
aSource: Kay, J. Intracellular protein degradation. Biochem. Soc. Trans. 6:789–797, 1978.
FIGURE 17.13 The ornithine decarboxylase reaction. The reaction uses a bound pyridoxal
phosphate group (PALP) that attaches to the α–amine of ornithine, leading to the release of the
α–carboxyl group and the formation of putrescine, one of the polyamines that serve to neutral-
ize the negative charge of DNA
The variation in the degradation rates is particularly significant when we consider that
proteins themselves are in a steady-state, just like intermediary metabolites. To appreciate
its significance for regulation, we conclude with an examination of a regulatory pathway for
protein synthesis.
17.6 THE mTOR PATHWAY IN THE CONTROL OF PROTEIN SYNTHESIS
The protein mTOR is a kinase that is central to the control of protein synthesis. It can be
regulated positively and negatively: insulin activates it, whereas AMP kinase inhibits it
(Figure 17.14).
mTOR is an abbreviation for mammalian target of rapamycin (Box 17.2). Insulin (and
other tyrosine kinase-mediated growth factors) leads to the activation of the kinase Akt. In
turn, Akt catalyzes the phosphorylation of the G-protein TSC (tuberous sclerosis complex,
named for a tumor that develops when the protein is dysfunctional). Inactivation of TSC
relieves mTOR inhibition, similar to how the inhibition of GSK3 relieves the inhibition of
glycogen synthase, another action of insulin (Chapter 13). AMP kinase is activated under
circumstances when the concentration of AMP increases, such as when increased ATP uti-
lization, that is not matched by ATP supply, leads to increased ADP concentration. ATP is
then regenerated through the near-equilibrium adenylate kinase reaction:
(17.3) 2 ADP ® ATP + AMP
When this happens, the concentration of AMP increases, activating AMP kinase and inac-
tivating mTOR.
mTOR catalyzes the phosphorylation of several targets including those controlling pro-
tein synthesis. For example, mTOR phosphorylates and activates an initiation factor in pro-
tein synthesis. Another mTOR phosphorylation target is S6 kinase (S6K), present in the
444 17.6 The mTOR Pathway in the Control of Protein Synthesis
FIGURE 17.14 Regulation of protein synthesis at mTOR. Insulin, via steps outlined in Chapter
13, leads to the activation of the kinase Akt (also called Protein Kinase B), which phosphorylates
and inactivates the G-protein TSC. This action lifts the inhibition that TSC exerts on mTOR, lead-
ing to the activation (phosphorylation) of S6K, a kinase that leads to increased protein synthesis.
Activation of AMPK (through increased concentrations of AMP), on the other hand, leads to the
phosphorylation and inactivation of mTOR, and the suppression of protein synthesis. Shown for
reference is another action of Akt: namely, the phosphorylation and inhibition of GSK3, which
lifts the inhibition of glycogen synthase.
small subunit of the ribosome (the S stands for small ribosomal subunit). Phosphorylation
of S6K promotes elongation in protein synthesis. The net effect of mTOR activation is the
stimulation of cellular protein synthesis.
Insulin stimulation of protein translation reflects the growth-promoting properties that
it shares with other cellular growth factors. AMP kinase suppression of protein translation
antagonizes growth and allows energy conservation by the cell. While we have seen that
several high-energy phosphate groups are directly consumed during protein synthesis, the
mechanism of energy-sparing is the diminished formation of the enzymes that catalyze
energy-utilizing pathways.
Due to the variation in protein degradation rates, inhibition of the overall rate of protein
synthesis does not affect proteins equally. Those with long half-lives will be little affected
by alterations in protein synthesis rates because the amount accumulated will take a long
time to be affected. Thus, while this means of regulation appears to be completely nonselec-
tive, it produces differential effects that can largely be read from a table of half-lives such as
Table 17.2.
Finally, all forms of regulation in cells and organisms can occur together. We may formally
divide regulation into short-term and long-term for experimental or conceptual convenience.
While translation can be considered another long-term effect, most cell regulation occurs at
the transcriptional level (see the chapter addendum). mRNA turnover, being relatively rapid,
allows the cell to produce precisely the amount of mRNA needed to drive protein synthesis,
based on its needs and sensitive to the control elements upstream of the gene in the DNA.
Changes in mRNA are tempered by protein translation short-term control mechanisms and,
most fundamentally, the rate of ATP utilization in the cell cytosol. All regulatory features
move us from one steady-state to the next.
SUMMARY
Protein synthesis requires the convergence of three types of RNA: ribosomal, messenger,
and transfer. The ribosome is the scaffold composed of a small and a large subunit, each of
which are complexes of proteins and rRNA. The small subunit forms a complex first, bind-
ing a charged (amino acid bound) tRNA, mRNA, and proteins serving as initiation factors.
With the addition of the large ribosomal subunit, the protein factors are released, and elonga-
tion ensues. Elongation factors assist the binding of charged tRNA to the ribosome, and the
peptide bond is formed. Termination occurs at a codon for which no corresponding tRNA
exists, and the completed protein is released. The formation of each charged tRNA requires
a distinct tRNA synthetase enzyme. Recognition of the tRNA on the mRNA at the ribosome
is due to a triplet anticodon sequence in tRNA base pairing with a three-nucleotide codon
in mRNA.
The correspondence of the codon to the amino acid is the genetic code. With the code,
an mRNA sequence can be converted to a unique protein sequence. Chaperone proteins
catalyze the folding of newly emerging proteins into their active configuration. Proteins
are also subject to various post-translational modifications that alter their properties, such
as phosphorylation and methylation. The process of translation is subject to regulation
in eukaryotes. For example, the heme-regulated inhibitor is a kinase that phosphorylates
a eukaryotic initiation factor, thus attenuating protein synthesis. mTOR is a kinase that
stimulates protein synthesis. mTOR is stimulated by insulin and inhibited by AMP kinase.
Proteins are degraded continuously in cells, in a process that occurs at distinct rates for dif-
ferent proteins. A major protein degradation route depends on the covalent incorporation of
a small protein called ubiquitin, followed by a protein complex breakdown, the proteasome.
Proteins are constantly synthesized and broken down; they exist at a steady-state within
cells.
REVIEW QUESTIONS
1. Tetracycline, an antibiotic used against bacterial infections, binds to the A site in

bacterial protein synthesis. Which phase of protein synthesis does this compound
specifically affect?
2. Several G-proteins are involved in protein synthesis. How is the action of these pro-
teins similar to G-proteins involved in hormonal regulation?
3. Why are there no examples of regulation of prokaryotic translation?
4. Leucine, one of the branched chain amino acids, is a potent activator of mTOR. Why
is this signaling appropriate?
5. While there is less control over proteolysis than protein synthesis, disease states are
associated with dysfunctional proteolysis. One example is Alzheimer's disease, in
which neural cells accumulate massive quantities of a protein called amyloid. Which
specific proteolysis system is likely responsible for this accumulation? If this is the
primary cause of the disease, what might be a possible avenue of treatment?
6. A few proteins are found to have amino acids other than the 20 we have considered
(known as the “canonical 20 amino acids”) that are incorporated translationally (i.e.,
not arising by modification after the protein is synthesized). One example is seleno-
cysteine, found in glutathione peroxidase. Would this amino acid have its own amino
acid synthetase? Its own codon? Does this make selenocysteine the “21st canonical
amino acid”?
446 Chapter 17 Addendum: Skeletal Muscle and Exercise
CHAPTER 17 ADDENDUM: SKELETAL MUSCLE AND EXERCISE
Skeletal muscle is energetically the most dynamic of tissues: ATP turnover can span a greater
range than any other cell type in the body, with the simple act of changing from rest to exer-
cise. Related to this switchover, the feeding–fasting and resting–exercise transitions were
considered briefly in Chapter 13. An extension to lipid metabolism was briefly considered in
Chapter 14. It is important to emphasize why lipid metabolism cannot maintain whole-body
glucose levels in fasting: fats cannot be converted to sugars. Just as the Krebs cycle can only
oxidize one substrate – acetyl CoA – the Krebs cycle cannot utilize its intermediates for net
glucose formation.
Maintaining blood glucose during periods of fasting requires gluconeogenesis. Since
the substrate for this glucose formation is largely amino acids, fasting requires proteoly-
sis; muscle is the major protein source. Analysis of muscle mass following weight-reduction
programs have shown that obese individuals do lose protein in addition to fat – as must
be the case. Surprisingly, the change in strength, measured as exercise performance, is not
considerable. In obesity, there is not only an excess of fat, but also of protein when compared
to lean individuals. However, exercise performance is relatively poor to begin with; this has
been categorized as “poor quality muscle”. It is likely that much of the musculature in obesity
is postural, to support a heavier frame.
Exercise is divided between two extreme types: aerobic and anaerobic. These correlate
to the two major muscle types: slow muscle, or Type I (having many mitochondria and using
lipids as fuel), and fast muscle, or Type II (having few mitochondria and using carbohydrate
as fuel). Each has a distinct response to exercise. For Type I, exercise signals act through
transcription factors that induce mitochondrial formation, and the pathway of lipid oxi-
dation. There is little appreciable gain in muscle mass. This exercise type corresponds to
aerobic exercise, such as cycling or running, although no exercise is purely one form or the
other. Increases in muscle mass involve Type II muscle, a result of anaerobic exercise, such
as weight-lifting.
For most protein alterations, transcription of mRNA and its attendant control systems are
of primary importance. However, as a general rule, translation is the primary regulatory site
when considering long-lived proteins. The major proteins of muscle are the contractile fibers,
actin and myosin, with exceptionally long half-lives.
There is ongoing proteolysis during muscle growth, but the rate of proteolysis is gener-
ally constant. The increase in muscle protein is triggered in part by the mTORC1 regulatory
system described in this chapter that increases the formation of the muscle proteins from the
existing mRNA. The increase in muscle mass – also known as muscle hypertrophy – has
another feature that has recently been uncovered: an increase in ribosomal RNA. The change
takes some time to appear, as the amount of rRNA is about 80% of the total cellular RNA,
so it can take weeks to observe an increase over this large background. Weight-training or,
more generally, resistance training can increase the total rRNA content of cells, increasing
the total capacity for translation. Resistance training is not only useful for strength building,
but also as a second component to a weight-loss program, as it adds muscle – and attendant
strength – as the body adapts to a new, healthier weight.
KEY TERMS
acceptor site
aerobic
anaerobic
anticodon
(catalytic) triad
chaperone
charged tRNA
codon
colloids
co-translational modifications
cyclosporine
genetic code
heme regulated inhibitor
immunophilins
mTOR
muscle hypertrophy
peptidyl site
pathway-irreversible
pathway-reversible
polycistronic
post-translational modifications
proteasome
resistance training
ribosome
ribozymes
S6 kinase
Shine–Dalgarno
signal sequence
svedberg
ubiquitin
wobble hypothesis
BIBLIOGRAPHY
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511–519.
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Biochem Sci 20 (1995) 105–108.
V.C. Figueiredo. Revisiting the Roles of Protein Synthesis During Skeletal Muscle Hypertrophy Induced
by Exercise. Am J Physiol Regul Integr Comp Physiol 317 (2019) R709–R718.
S. Joanisse, C. Lim, J. McKendry, J.C. McLeod, T. Stokes, S.M. Phillips. Recent Advances in
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Appendix
A1. MATHEMATICAL IDEAS
A1.1 VECTORS
Many common entities have only a magnitude: speed, weight, and density are examples. Others require both a magni-
tude and a direction to capture their essence: these are vectors. For example, a car’s speed can be represented as 30 mph,
but a description of its velocity needs more, such as 30 mph north.
Our first encounter with a vector in this text is in the concept of polarity. The magnitude of this vector depends on
the relative attraction for the binding electrons between the two nuclei. The direction is from the lesser to the more
electronegative atom. When we add these vectors (to determine molecular polarity), we must take into account both
magnitude and direction. Vector addition can be solved graphically (Figure A.1), after rearranging the vectors A and B,
so that their origins coincide, retaining the same direction and length as the original vectors. This construction pro-
duces the vector sum as shown in the figure. Note the magnitude of the sum – C is (in general) distinct from the sum of
the magnitudes of A or B. If A and B are exactly 180 degrees apart and of equal magnitude, the vector sum is zero; this
is the case of the net polarity of CO2.
A second important case of vectors is flow of protons in mitochondria, bacteria, and chloroplasts establishing the
proton-motive force, the basis of energy production. In this case, we did not consider vector mathematics explicitly; only
the directional traversal of the membrane is essential.
A1.2 LOGARITHMS
The hierarchy of operations in mathematics can be written as:
I. Addition and its inverse, Subtraction.

II. Multiplication and its inverse, Division.
III. Exponentiation and its inverse, Logarithms.
Level I is the familiar process of addition. Even if this was the only operation available, we could still perform operations
that usually employ levels II and III, but it would be tedious. A step up from addition, multiplication effectively tallies
objects by grouping them into multiples. For example, rather than add 300 individual pencils, we can arrange them into
groups of 10; finding 30 of these means 30 multiples of 10.
For the Level III, consider bacterial growth: each cell divides, each of those cells divide, and so forth. This is exponen-
tial growth. For three events, rather than multiplying a number by itself repeatedly, say 2 x 2 x 2, we can abbreviate this
by writing 23. The superscripted 3 here tells us how many times we have multiplied 2 by itself. The 2 is called the base
and the 3 is the exponent or power. This can be expressed as “two to the third power”. The interesting property of expo-
nents is that we can add the exponents themselves rather than just carrying out the whole operation. We thus could say:
(A.1) 23 * 2 4 = 27
which is easily verified by multiplying out both sides.

Now consider the inverse function of exponentiation: the logarithm (or just log). In our example of exponents, the
base 2 raised to the power 3 produces the answer 8. Then the log of 8 to the base 2 is 3.
We are usually interested in just a few bases: 2, 10, and e. The latter is the base of the natural logarithms. The ratio-
nale for e is somewhat far afield of this treatment; it is easily understood from the operations of calculus (the derivative
of the function, ex, is equal to the very same function, ex; thus, e is the base of the most important recursive function).
449
450 Appendix
FIGURE A.1 Vector addition. Slide the two vectors in the plane of the page, maintaining their
direction and magnitude, until their origins coincide. Construct parallel segments (dashed lines)
to each line and complete the parallelogram. The sum is the arrow from the common origin to
the intersection of the constructed (dashed) lines.
Base 2 is commonly used in measuring population growth. We use base 10 in pH discus-

sions; in fact, the pH is, itself, a base 10 logarithm of hydrogen ion concentration (actually its
reciprocal), and it’s useful because the concentration of hydrogen ions is very low in solution.
Thus, we can represent the concentration of hydrogen ions of 10−7 as simply a log form; we
say “it is a pH of 7”.
As logarithms can be viewed as picking off the exponents, and because exponents as we
have just seen are additive, this also must be true of logarithms. Hence the rule:

(A.2) log a * b = log a + log b
which is used in the derivation of the Henderson–Hasselbalch equation.

Two important points must be noted about logarithms. First, there are no units. This
means that the phrase “pH units” has no meaning. The hydrogen ion concentration has units,
but once we take its logarithm, we are extracting the exponent, producing a unitless number.
Secondly, the value of a change in pH is not a constant difference. It depends on the range.
In the study of chemiosmosis, we commonly encounter expressions that employ ΔpH as part
of the electrochemical potential. However, this is an inexact quantity; clearly the same ΔpH
starting from say pH 8 must be an order of magnitude greater than one starting from pH
9. Thus, logarithmic transformation can complicate understanding as it makes exponential
changes appear linear and eliminates the units of measure.
A1.3 GEOMETRIC MEAN
An uncommon method of taking an average, this arises prominently in the calculation of

isoelectric points in amino acids. The definition of pI for an amino acid with no additional
dissociable group (i.e., an extra acid or base) is:
(A.3) pI = ( pK1 + pK 2 ) /2
While this seems like a straightforward arithmetic mean, it isn't. We are dealing with loga-
rithmic expressions, so arithmetic means do not apply. We need what is known as the geo-
metric mean, where the values are multiplied before dividing by two, rather than being
added. This makes sense when we realize that this must be the case for finding an average
value for equilibrium constants: these are always related by multiplication in cases of con-
nected equilibria with polyprotic acids, like inorganic phosphate.
The rationale for using the geometric mean is that combining equilibrium constants
for sequential reactions involves multiplicative rather than additive operations. This goes
back to the original derivation of an equilibrium constant for an equation: the products
multiplied together divided by substrates multiplied together. It should be expected that a
combination of two reactions in series is also summarized by an overall equilibrium con-
stant that is the multiple of each individual constant. Thus, in forming the isoelectric point,
what appears to be an arithmetic average is masked by the fact that what is being added are
log values.
Appendix 451
A2. CHEMICAL FUNDAMENTALS
Even after taking two years of chemistry, students entering biochemistry are often unclear
about some elementary chemical ideas. In part, this is due to the enormous amount of infor-
mation presented, and the difficulty in knowing what is essential and what is not.
Another factor that hinders understanding starts at the beginning of a modern chemical
education: presenting the periodic table as based on C having a relative weight of 12. This is
technically true, but only of real importance to the Bureau of Weights and Measures. It has
no functional significance.
In fact, we should think of the periodic table as based on hydrogen, the lightest element,
assigned the relative weight of 1. All other elements are multiples of H. Experimentally, C
is 12 times the mass of H, and so C has a molecular weight of 12. Oxygen has a molecular
weight of 16. The mole (abbreviated mol) is the number of atoms in 1 gram (g) of H: that
number is 6 x 1023 (Avogadro’s number). Thus, 12 g of C or 16 g of O contain the same number
of atoms as 1 g of H.
A subtlety of Avogadro’s number is that its units are themselves variable. For example, it
can be applied to a number of atoms or molecules. Usually, we consider a mol as an Avogadro
number of an entity. Take the connected collection of atoms that is glucose, C6H12O6. This
entity has a molecular weight of 180 g. This means that 180 g of glucose is equal to 1 mol
of glucose. In what we can call chemical arithmetic, a reaction can be considered to follow:

(A.4) 1 mol of A + 1 mol of B ® 1 mol of C
for a joining reaction. This adds up in the usual mathematical manner only when we take
into account the actual weights of each term. Otherwise, abstracting the numerical part of
Equation A.4 without units would produce: 1+1 = 1!
Protons and electrons have an opposite charge and attract; this notion is a primitive fact of
chemistry (that is, we don't know why it is true since charge itself is a mystery). It is the origin
of all electrical phenomena. The neutrons have no charge but co-exist in the nucleus for the
purpose of atomic stability. This has no bearing on the chemistry that we consider, but it is
interesting to note that without neutrons, protons could not possibly exist so close together.
The mutual electrostatic repulsion would cause them to fly apart. Protons and neutrons are
attracted to one another by another force that acts at very short range and overcomes the
proton–proton electrostatic repulsion. The approximate balance of these forces explains why
most elements have a similar number of protons and neutrons, making the atomic mass
roughly double the atomic number. The larger elements have an excess of neutrons over pro-
tons, which stabilizes the nuclei.
The electron arrangement outside the nucleus is all-important, and is a balance between
two electrostatic influences. From the standpoint of the atom, there is the attraction between
proton and electron, which keeps the electrons within the orbit of the nucleus. Broadly speak-
ing, there are two types of electrons: those that we call valence electrons – and the rest. Only
the valence electrons are considered to interact with other atoms, as they are the furthest
from the nucleus and thus less attracted by the positive charge. There are other subtleties. For
example, the outer electrons also are shielded by the inner electrons from the charge of the
proton and, thus, experience a reduced electrostatic attraction. The non-valence orbitals are
important in ions such as Fe2+, which form coordinate bonds to ligands like heme; the inner
orbitals of Fe2+ allow it to have six binding positions.
Electrons do not actually orbit the nucleus like the planets around the sun. Despite this,
the volume of space that is occupied by the electrons is called an orbital. The actual shape of
orbitals (such as the first one appearing in the text for water) is calculated from probability
considerations based on quantum mechanism, developed in the early 20th century. While
there is some complexity to quantum chemistry, the results are extremely simple: the orbit-
als are probability clouds in which there is a high probability of finding particular electrons.
Electrons associated with atoms have a regularity that matches the types of orbitals that
exist within them. In order, they are called s, p, d, f, etc. The atoms we are concerned with
usually have electrons only in the first few – s and p – and only occasionally the d orbitals.
452 Appendix
Each individual orbital can have up to two electrons in it. The s orbital is spherical and can
contain a maximum of two electrons. There are three p orbitals, each with a maximum of
two electrons, so that p orbitals collectively can hold up to six electrons. The d orbitals are a
set of five, each with two electrons, so that these can accommodate up to ten electrons. Each
succeeding atom has just one more proton than the preceding one, and is also accompanied
by one more electron. For hydrogen, there is just one proton and one electron; the electron
occupies an s-orbital, which is spherical. Carbon has six electrons, two of which are in an
s-orbital, and the other four in p-orbitals. The p orbital shapes resemble a dumbbell with zero
electron density in its center. The three p orbitals are at right angles to each other.
Electron configurations of atoms have a symbolism that includes the orbitals as well as the
number of electrons in each orbital, along with a further number preceding these which is
the principal quantum number (n), roughly where the electron is with respect to the nucleus,
or its “shell”. Alternatively, we can think of this as the electron’s energy level. The values of n
can be mapped to the periodic table: the first row corresponds to n = 1, the second to n = 2,
and so forth. The notation for H is 1s1, which means n = 1, the electron is in the s orbital, and
the superscript 1 means there is one electron. A further mapping to the periodic table is that
the orbitals correspond to column blocks in the periodic table. The first two columns are the
s-block, the six columns on the right-hand side are the p-block, and the ten columns in the
center are the d-block.
The C atom has 2 electrons in an inner s orbital (core electrons, not involved in bonding),
and 4 valence electrons (i.e., bonding electrons); its electron configuration is [1s2]2s22px12
py12pz0. Here, the electrons not involved in bonding to other atoms are enclosed in square
parentheses – the core electrons – to separate them from the electrons that are involved in
forming bonds – the valence electrons.
A molecule is a collection of atoms that are covalently linked. This covalent linkage means
that orbitals from two atoms overlap and the electrons are shared between nuclei. This join-
ing of atoms creates molecular orbitals. Like the atomic orbitals just considered, these orbit-
als also can hold only a maximum of two electrons each. All four C–H bonds in methane are
identical, and yet the electrons involved are from s and p orbitals in C and from s orbitals in
H. The model of hybridization of atomic orbitals to form molecular orbitals can account
for the experimentally observed bonding in carbon compounds. For example, the bonds in
methane are known to be identical. To understand the hybridization of methane, imagine
that one of the 2s electrons of C is promoted to the 2p level. This would produce a configura-
tion of [1s2]2s12px12py12pz1. We pair this with a single s electron from H. The valence electrons
are said to form an sp3 hybrid. Each orbital has one electron and all four orbitals are equiva-
lent. We summarize the situation by saying that CH4 has a structure in which the C–H bonds
are sp3 hybridized. The bonds themselves are known as sigma or σ-bonds, and concentrate
electron density between the C and H nuclei.
While this is obviously a rough view of electronic bonding, it is useful in understanding
chemical behavior. Because the electrons in each C–H orbital repel the electrons in the other
C–H orbitals, they are arranged in space so that they are the maximal distance apart. This
produces the structure of the methane molecule and other tetrahedral carbon compounds.
A distinct type of hybridization is need to explain double bonds. In the example of ethylene
H H
C C
H H
it is clear that there are only three connections made by each carbon, and that two electron
pairs combine the two carbon atoms. The hybridization model applied to this situation again
starts with the C ground state configuration of [1s2]2s22px12py12pz0. Once again, we start by
imagining the promotion of an electron from the 2s2 orbital: [1s2]2s12px12py12pz1. For this
situation, however, we combine just two of the p orbitals with the s orbital, resulting in sp2
molecular orbitals. Each C forms two sp2 bonds between an H and one sp2 bond to the other
C. These are all sigma bonds, and all have the same length.
Appendix 453
FIGURE A.2 The π bond. The dumbbell shapes represent individual p-orbitals; their edge-
wise overlap is the π bond.
FIGURE A.3 Curved arrow notation. (a) For reactions (b) For resonance forms.
We have one p orbital left over on each carbon, with an electron in each: the pz orbital.
These two are oriented perpendicular to the plane of the rest of the molecule, as illustrated
in Figure A.2. The electron density in both halves of a dumbbell type structure. The orbital
overlap of these is on the edge rather than directed between the nuclei of the atoms. This
sideways overlap of p orbitals is known as a pi (π) bond. There are 2 electrons in the π bond.
As there is already a sigma bond between these two carbons, we call this a double bond,
although it is clear that the double bond is constructed of two different types of covalent
linkage. The greater reactivity of double bond is due to the electrons of the π bond.
A2.1 CURVED ARROW CONVENTIONS
Curved arrows are symbols superimposed on molecular structures to suggest how electrons
might move to explain two separate processes: reaction mechanisms and resonance. The
convention for the arrows is that the tail originates from an electron pair, and the arrowhead
points to a relatively electron deficient destination. The convention is a rationale of of elec-
tron movement in chemical mechanisms for organic chemistry.
The first situation is illustrated in Figure A.3a. Here, a reaction is taking place between the
lone pair of electrons on the primary amine and the partially positively charged carbon of a car-
bonyl group. The reaction product is a doubly charged intermediate that we can imagine after
the electron rearrangement. The movement of the electrons is implied as following the arrows.
The second situation is illustrated in Figure A3b. Here, the electron pair relocates from
the π bond of the carbonyl to the C–N bond, and a separate resonance form is shown (a
zwitterion, just like the one of Figure A3a). However, unlike reactions, resonance forms are
not individual structures that result from electron rearrangement. Resonance structures are
separate representations of a molecule that cannot be depicted by one diagram. Therefore,
the meaning underlying the curved-arrows for resonance are distinct for resonance struc-
tures; they only suggest how an arrangement of one representation could lead to another.
A3. DERIVATION OF THE GENERAL FREE ENERGY EQUATION
Changes in enthalpy, entropy, and free energy at standard state are indicated with a super-
script degree symbol appended to their abbreviation: ΔHo, ΔSo, and ΔGo, respectively. The
standard free energy change (ΔGo) is of particular interest, and can be related to the equilib-
rium constant (Keq). To show this relationship, we begin with the following equation:
(A.5) DG = DGo + RT ln [C ][ D]/ [ A ][B]

454 Appendix
This equation describes the reaction:

(A.6) A + B C + D
Equation (A.5) originates from free energy equations for each reaction component, such as:
(A.7) GA = GA o + RT ln [ A ]
The logarithmic relationship between free energy and concentration of the component is the
result of a derivation that uses the ideal gas law. The logarithmic term arises from an integra-
tion of a reciprocal pressure, which is then related to concentration. Perhaps surprisingly, the
resulting equations have wide applicability to situations which are not ideal, and to liquids as
well as gases. Using equations similar to Equation A.7 for the other components of the reac-
tion (Equation A.6) and the linear free energy relationship:
(A.8) G A + GB = G C + B D
The final equation (Equation A.5) results from collecting terms.
A4. LIPIDS
A4.1 FATTY ACIDS
Table A.1 shows a more extensive listing of fatty acids, along with their structures and melt-
ing points. The latter show a regular increase with chain length for the unsaturated fatty
acids. Table A.1 also shows that double bonds profoundly decrease the melting points.
A4.2 PHOSPHOLIPIDS
A listing of phospholipids along with their structures is shown in Figure A.4.
A5. DERIVATION OF EQUATIONS FOR REVERSIBLE ENZYME INHIBITION
Reversible inhibitors add one or more equilibria to the enzyme forms of the mechanism.
Each of the three common cases is considered in turn.
TABLE A.1 Fatty Acids

Identity Common Name Systematic Name Structure Melting Point
Saturated 4:0 butyric butanoic CH3(CH2)2C02H −5.5
6:0 caproic hexanoic CH3(CH2)4C02H −4
8:0 caprylic octanoic CH3(CH2)5C02H 16
10:0 capric decanoic CH3(CH2)0C02H 31
12:0 lauric dodecanoic CH3(CH2)10COOH 44
14:0 myristic tetradecanoic CH3(CH2)12COOH 52
14:0 palmitic hexadecanoic CH3(CH2)14COOH 63
18:0 stearic octadecanoic CH3(CH2)16COOH 70
Unsaturated
16:1(d9) palmitoleic hexadecanoic CH3(CH2)5CH=CH(CH2)1C02H 0
18:1Wl oleic octadecanoic CH3(CH2)1CH=CH(CH 2)1COOH 16
18:2(d9·12) linoleic octadecadienoic CH3(CH2)4(CH=CHCH2)2(CH2)6COOH −5
18:3(d9,12,15) linolenic octadecatrienoic CH3CH2(CH=CHCH2)3(CH2)6COOH −11
20:3(d5,8,11,14) arachidonic eicosatetraenoic CH3(CH2)4(CH=CHCH2)4(CH2)2COOH −50
Appendix 455
FIGURE A.4 Phospholipids.
A5.1 COMPETITIVE INHIBITION
By definition, the inhibitor I binds to the free enzyme E at equilibrium, defined in the direc-
tion of dissociation,
(A.9) EI E + I
(A.10) K is = ([E]*[I]) /[EI]
The presence of the new enzyme form EI requires a new conservation equation:
(A.11) [E]tot = [E] + [ES] + [EI]

456 Appendix
Because EI is a dead-end complex, this reduces the concentration of the enzyme present in the
other forms and must therefore decrease the initial velocity. The derivation is similar to the
uninhibited case, except that the presence of the extra enzyme form means that Equations
(A.10) and (A.11) are used in order to eliminate all enzyme terms from the equation apart
from ES; otherwise, the derivation proceeds as before. Rearranging Equation (A.10):

(A.12) [EI] = ([E]*[I]) /Kis
Substituting Equation (A.12) into Equation (A.11):
(A.13) [E]tot =[E]+[ES]+ ([E]*[I]) /Kis

Rearranging and solving for [ES] as before, with the same definition of Vmax produces the
final equation:

(A.14) { }
v = Vmax [S] / K M (1 + [ I]/K is ) + [S]
A5.2 ANTICOMPETITIVE (UNCOMPETITIVE) INHIBITION
The derivation for the anticompetitive case is similar to that of competitive inhibition, except
that the extra equilibrium involves the ES complex:

(A.15) ESI ES + I
(A.16) K ii = ([ ES] *[ I]) / [ ESI]
The conservation equation then becomes:

(A.17) [E]tot = [E] + [ES] + [ESI]
The derivation precedes as before: these new equations are used to first eliminate the [E]
term and then solve for [ES]. The final equation for anticompetitive inhibition is:
(A.18) {
v = Vmax [S]/ K M + [S](1 + [ I]/K ii ) }
A5.3 MIXED (NONCOMPETITIVE) INHIBITION
In the final case, we have a mixture of competitive and anticompetitive inhibition. Both
equilibria already introduced are present; both E and ES reversible bind to I. The conserva-
tion equation is:

(A.19) [E]tot = [E] + [ES] + [EI] + [ESI]
and the resulting equation for mixed inhibition is:

(A.20) { }
v = Vmax [S]/ K M (1 + [ I]/K is ) + [S](1 + [ I]/K ii )
Appendix 457
FIGURE A5 Kinetics of enzymes at lipid-water interface. Compared to a water soluble reaction
like acetylcholine esterase, the kinetic curve for the interfacial acting enzyme phospholipase A2
has two regions. Below the critical micelle concentration (CMC), the enzyme resembles a nor-
mally saturated reaction. When the concentration of substrate reaches CMC, the velocity rises
sharply with substrate concentration as a new surface is created for the enzyme to bind to.
A6. THE LIPID-WATER INTERFACE
Enzymes with lipid substrates are often themselves water soluble, but act at the lipid-water
interface. Because most of our thinking about enzyme action assumes that all reaction
components are in the same aqueous phase, a departure from this viewpoint is required to
understand interfacial catalysis.
The concept of interfacial catalysis is already implicit in the operation of membrane trans-
porters, which take a substance from one aqueous phase, pass it through the membrane,
and deposit it into a separate aqueous phase. However, this situation requires no new kinetic
treatment; transport kinetics are indistinguishable from enzyme kinetics.
With lipid enzymes that act at an interface, the situation is different. We will consider the
example of phospholipase A 2, the enzyme most commonly used for analysis of the general
problem of lipid-water interface. Figure A.5 compares the kinetics of hydrolysis of phospholi-
pase A2 with an esterase enzyme, such as acetylcholine esterase. Notice the unusual shape of
the phospholipase catalyzed reaction: rather than a monotonic increase, there is a small rise
in activity that nearly levels off, and then at a point marked CMC, an abrupt rise in velocity
until a saturation point is reached.
This curve can be explained in the following way. The first region, below CMC, represents
Michaelis–Menten type kinetics in which individual substrate molecules interact with the
enzyme and it becomes nearly saturated in the typical way. However, once the lipid mol-
ecules reach the point where a cluster is formed – the critical micelle concentration (CMC) –
the reaction shifts to essentially a new, secondary enzymatic activity. This is because the
substrate has changed. Rather than being an individual lipid molecule, it is now in the form
of a micelle. Now the enzyme binds a new surface and has far greater activity. Thus, the figure
can be viewed as two separate, sequential, saturation curves.
Index
5′ end, 433 levels of protein structure, 93–99

oxygen binding, 100–103
A peptide bond, 90–92
peptides and proteins, 92–93
α-carbon, 79 R groups
Acceptor stem, 429 functional groups, 81–85
Acetone, 336 polarities, 80–81
Acid–base catalysis, 131–132 Amino end, 91
Acid–base properties and charge Ammoniotelic, 400
α-carboxylate and α-amine groups Amperes, 175
electric field effect, 88–90 Amphiphiles, 41
inductive effect, 87–88 Anabolic steroids, 350
multiple dissociable groups, 90 Anaerobes, 364
titration and net charge, 85–87 Anaerobic exercise, 295, 446
Acid–base reactions, 6–7 Anaplerotic, 229
Acidic phospholipid pathway, 343 Angiotensin II, 92
Acids and bases, 26–27 Annihilation, 247
Aconitase, 218–220 Anomeric carbon, 59
Activation energy, 111 Anomeric effect, 62
Active site, 111 Antennae, 270
Acyl carrier protein, 341 Anticodon site, 429
Acyl transfers Anticompetitive (uncompetitive) inhibition, 120, 122–124, 456
carnitine, 147–148 Antiparallel beta sheet, 94, 96
CoA, 146–147 Apolipoprotein, 325
lipoic acid, 148 Apoptosis, 262
Adenosine 5′-triphosphate (ATP), 170–171 Aquaporins, 50
and ADP, 180 Archaea, 11
binding of, 188 Arginine, 377–378
energy coupling Arrhenius definition, 7
adenylate kinase, 174 Arsenate poisoning, 203–205
in asparagine synthetase, 171–172 A site, 433
creatine phosphokinase, 173 Assays, 105, 113–114
NDP kinase, 173–174 Assimilation, 363
PFK, 191 Atomic charged forms, 108–109
synthesis, 247–249 Autophagy, 442
Adenylate kinase, 98, 174 Auxotrophic, 265
Adipocyte, 10
Adrenergic receptors, 301 B
Aerobic exercise, 446
Affinity, 120 Bacteria, 11
Affinity chromatography, 106 Beer’s Law, 113
Agar, 70 Beriberi, 140
Agarose, 71 Beta form, 61
α-helix, 93 Bile, 325
α-hydrogen, 79 Bile salts, 47
Alanine, 376 Binding change, 248
Aldolase, 193–194 Biochemical redox potentials, 178
Aldoses, 55 Biochemistry, origins of, 1
Alkaloids, 398 Biological systems, 12
Allopurinol, 392 Biopterin cofactors, 374
Allosteric enzymes, 126–128 Biotin, 149–150
Alpha form, 61 Bisphosphonates, 360
Alpha helix, 95 Bohr effect, 103
Alveoli, 47 β-oxidation, 358
Amide, 90 Branched polysaccharides, 68
Amino acids Branch point, 68, 290
acid–base properties and charge (see Acid–base properties and Bronsted definition, 7
charge) Brown adipose, 39
biology of, 79–80 β-sheet, 93
common structure, 79 Buffering range, 31
459
460 Index
Buffering region, 30, 85 Concerted mechanism, 131

Bundle sheath cells, 282 Condensed phase water, 18–20
Consensus sequences, 419
C Cooperativity, 102
Coordinate covalent bond, 100
C3 plants, 282 Coordination number, 101
C4 plants, 282 Copper, 155
Calmodulin, 97 Coproducts, 139
Calvin cycle Core complex, 419
energy consuming portion, 277 Cori cycle, 305
GAP, 277–282 Corrosive gas, 363
ribulose bisphosphate carboxylase, 275–277 Cosubstrates, 139
segment, 317 Co-translational modifications, 437–439
CAM plants, 283 Coulombs, 176
Capping, 415 Coulomb’s law, 33
Cap structure, 421 Coupling, 253
Carbohydrates Covalent bonds, 19
carbohydrate derivatives, 68–73 Crassulaceae, 283
disaccharides, 62–63 Creatine phosphokinase, 173
monosaccharides, 55–59 Critical micelle concentration (CMC), 45
polysaccharides, 64–68 Current, 175
ring formation in sugars, 59–62 Cyclooxygenase, 351
Carbohydrate synthesis reactions, 286 Cytochrome c, 245
Carbon fixation, 268–269, 286 Cytochrome P450, 349
Carbon reactions, 209, 265 Cytosol, 11
Carboxylation
biotin, 149–150 D
vitamin K, 150–151
Carboxyl end, 91 Dark reactions, 265
Carnitine, 329 Dehydrogenases, 132
Catalytic rate constant, 115 Denitrification, 363
Catalytic triad, 131 Deoxy sugars, 70
Cell surface binding proteins, 104 Dielectric, 32–34
Cell theory, 9–11 Dielectric constant, 24
Cellulose, 66 Diester, 42
Ceramide, 353 Dietary essentials, 156
Chaperone proteins, 100, 445 Diffusion, 50
Charge, 175 Dipole moment, 24
Charged capacitor, 33 Disaccharides, 62–64
Charged tRNA, 431 DNA gyrase, 412
Chiral carbon, 56 DNA ligase, 415
Chiral molecule, 56 DNA polymerase, 412
Cholesterol, 42–44 DNA repair
metabolism, 346–351 excision repair, 416
Choline, 42 mismatch repair, 415–416
Chromatography, 105 PARP, 416
Chylomicron, 325 DNA replication
Chyme, 325 double helix, 414–415
cis-aconitate, 220 in eukaryotes, 415
Citrate synthase, 217–218 initiation, 412–413
Citric acid, 6, 211 primer formation, 414
Citric acid cycle, 211 replisome, 413–414
Clustered regularly interspaced short palindromic repeats Domains, 94–97
(CRISPR), 425 Double bond, 453
Cobalamin, 332, 380 Double helix, 423
Cobalt ions, 155–156 Down regulation, 300
Codon, 432 Downstream, 418
Coenzymes
acyl transfers (see Acyl transfers) E
bound and mobile, 139–140
carboxylation (see Carboxylation) EF hand domain, 97
defined, 139 Eicosanoids, 40
exchange coenzymes, 151–154 Electrical potential, 176
redox coenzymes (see Redox coenzymes) Electric circuit analogy, proton gradient, 250–251
and vitamins, 140 Electric field, 87
Collision theory of reaction rates, 3 Electric field effect, 88–90
Colloids, 432 Electrochemical cell, 175
Competitive inhibition, 120, 122, 455–456 Electrodes, 176
Complementarity, 407 Electrogenic, 252
Complete pathway, 199 Electronegative, 8
Index 461
Electronegativity, 17 Exons, 422

Electron releasing, 88 Exonuclease, 415
Electrons, 451–452 Exothermic, 164
Electron sink, 141, 194 Extracellular binding proteins, 104
Electron transport chain, 235 Extremophiles, 12, 426
Electron-withdrawing, 88
Electrophoresis, 107 F
Elongation, 417
Emulsification, 44, 325 Faraday, 176
Enantiomers, 56 Fat cells, 49
Endoplasmic reticulum, 11 Fatty acid biosynthesis
Endothermic, 164 carboxylation of acetyl-CoA, 339–340
Energy, 8–9 export of acetyl-CoA, 336–339
free energy derivation, 182–183 two-carbon fragments to form palmitate, 340–343
internal energy, 161 Fatty acid oxidation
mobile cofactors, 180–181 activation, 327–328
of redox reactions β-oxidation, 330–334
electricity fundamentals, 175–176 transport, 328–330
electrochemical cell, 176–177 Fatty acids, 454
reduction potentials, 179–180 cis and trans fatty acids, 37
reduction potentials and free energy, 178–179 defined, 35
standard reduction potentials, 177–178 delocalized, 37
Energy balance, 207, 227–228 isolated and conjugated double bonds, 37
Energy-linked transhydrogenase, 251 isolated double bonds, 37
Enolase, 196–197 localized, 37
Enthalpy, 8, 22 parts of, 36
Entner–Doudoroff pathway (ED), 209 polyunsaturated, 37
Entropy, 8, 22 properties of, 36
Enzyme complex, 213 saturated, 35
Enzyme conservation, 115 unsaturated, 35
Enzymes Feed-forward activation., 198
acid–base catalysis, 131–132 Fermentation, 200
allosteric enzymes, 134 Ferredoxin (Fd), 271
categories, 132 Ferritin, 104
defined, 111 Fischer projection, 55
double-reciprocal/Lineweaver–Burk plot, 125–126 Flavin adenine dinucleotide, 145
enzyme assay and initial velocity, 113–114 Flavin coenzymes, 144–146
enzyme-catalyzed reactions, 111–113 Flavin mononucleotide, 145
irreversible inhibition, 128–129 Fluoroacetate poisoning, 223
kinetic mechanism Fractional saturation, 101
assumptions, 115 Free energy, 9, 22, 165, 182
Michaelis–Menten equation, 115–117 Fructose bisphosphatase, 279
membrane transport proteins, 132–133 Fructose metabolism, 205–207
nucleophilic substitution, 129–131 Fructose-2,6-P2, 191, 192
reversible inhibition Fumarase, 226
anticompetitive inhibition, 122–124
competitive inhibition, 120–122 G
mixed inhibition, 124–125
Epidermal cells, 10 Galactose utilization, 318–320
Equilibrium Gas–liquid chromatography, 106
defined, 3 Generic reaction, 2
equilibrium condition, 4 Genetic code, 445
forward and reverse rates, 3, 4 Geometric mean, 450
Le Chatelier principle, 5 Glucagon, 296
Equilibrium balance, 112 Glucogenic, 372
Erythrocyte, 10 Glucokinase, 189
d-Erythrose, 58 Gluconeogenesis, 289
Essential fatty acids, 351 fructose-1,6-P2 to fructose-6-P, 309–310
Ethanol formation, 200–201 glucose-6-P to glucose, 310–311
Eukarya, 11 lactate dehydrogenase, 304–305
Evolution pathway integration
defined, 11 feeding–fasting transition, 311–312
species hierarchy and, 11–12 resting–exercise transition, 312–313
Exchange coenzymes pyruvate to PEP, 305–306
pyridoxal phosphate, 152–154 transport of oxaloacetate, 306–309
thiamine pyrophosphate, 151–152 Glucose-6-phosphatase, 310
Excision repair, 415, 416 Glucose phosphate isomerase, 189–190
Exciton transfer, 270 Glucose transport, 185–188
Exergonic, 166 Glutathione, 394
Exome sequencing, 423 Glyceraldehyde phosphate dehydrogenase, 194–195
462 Index
Glycogen, 68 I
Glycogen branching enzyme, 290
Glycogenin, 290 Imidazole, 81
Glycogen metabolism Imidazole equilibria, 84
glycogenolysis, 291–296 Immunophilins, 439
glycogen synthesis, 289–291 Induction, 87
physiological context of, 296 Inductive effect, 87–88
regulation of Initial velocity, 114, 115, 135
AMP kinase, 303–304 Inorganic chemistry, 2
epinephrine, 299–300 Inorganic phosphate, 41
glucagon, 296–299 Insulin, 300
insulin, 300–303 Insulin receptor substrate 1 (IRS-1), 301
Glycogen phosphorylase, 291, 294 Insulin-resistant, 357
Glycogen phosphorylase kinase, 299 Intermediary metabolism, 289
Glycogen storage diseases, 295 Internal energy, 8, 161, 162
Glycogen synthase kinase 2 (GSK-2), 304 Intestinal mucosa, 187
Glycogen synthase kinase 3 (GSK-3), 300 Intracellular binding proteins, 104–105
Glycolysis Intrinsic factor, 382
aldolase, 193–194 Introns, 422
alternative endpoints, 208 Inverted micelles, 52–53
alternative entry points, 207 Ionic bonding, 23
arsenate poisoning, 203–205 Iron, 155
energy balance and glycolytic connections, 206–207 Irreversible inhibition, 128
enolase, 196–197 Isocitrate DH, 223–224
ethanol formation, 200–201 Isoelectric point, 90, 107
fructose metabolism, 205–207 Isomerase enzymes, 132
glucose phosphate isomerase, 189–190 Isoprenes, 359–360
glucose transport, 185–186 Isozymes, 189
glyceraldehyde phosphate dehydrogenase, 194–195 Itaconate pathway, 231–232
glycolytic intermediates, 207–208
hexokinase, 186–189 K
lactate formation, 200
pathway thermodynamics, 201–202 Keto-enol, 198
phosphofructokinase, 190–193 Ketogenic, 372
phosphoglycerate kinase, 195 Ketone bodies, 334–335, 336–337
phosphoglycerate mutase, 195–196 Ketoses, 55
pyruvate kinase, 197–199 Kinetic constants, 117
red blood cell shunt pathway, 203 Kinetics, 2
triose phosphate isomerase, 194 K m, 119–120
Glycolytic intermediates, 207–208 Krebs cycle
Glycosidic bond, 62, 75 aconitase, 218–220
Glyoxylate cycle, 231 cis-aconitate, 220
Gout, 392 citrate synthase, 217–218
Grana, 266 cyclic pathway, 211–212
GroEL segment, 100 energy balance, 227–228
fluoroacetate poisoning, 223
fumarase, 226
H glyoxylate cycle, 231
Hairpin loop, 420 isocitrate DH, 223–224
Haldane relationship, 136 itaconate pathway, 231–232
Haworth Projection, 59 α-ketoglutarate DH complex, 224
Heat, 8, 161 malate DH, 226–227
Helix breakers, 94 of metabolic pathways, 229
Heme, 100 prochirality, 220–221
Heme regulated inhibitor (HRI), 437 regulation, 228–229
Hemiacetals, 59 R/S system of nomenclature, 221–223
Hemimethylated, 415 substrate of, 212–215
Henderson–Hasselbalch equation, 28–30 succinate DH, 225–226
Hexokinase, 186–189 succinyl-CoA synthetase, 224
High energy electrons, 9
High energy molecules, 9 L
High energy phosphate, 9
High heat retention, 24–25 Lac operon, 419
High-pressure liquid chromatography (HPLC), 106 Lactate formation, 200
Histidine, 377–378 Lactose intolerance, 65
Histones, 420 Lafora disease, 322
Holistic, 12 Laforin, 324
Hybridization, 452 Lagging strand, 413
Hydrogen bonding, 19, 96 Leading strand, 413
Hydrolase enzymes, 132 Le Chatelier principle, 5
Index 463
Lecithin, 44 enzyme behavior, 117–119

Leghemoglobin, 364 historical perspective of, 116
Leukotrienes, 352 kinetic constants, 117
Lewis definition, 7 mathematical form and scientific interpretation, 118
Ligase enzymes, 132 steady-state assumption, 115
Light reactions, 265 Midpoint potential, 179
Lipid(s) Minor groove, 409
cholesterol, 42–44 Mismatch repair, 415–416
composition, 40–41 Mitochondria, 11
droplets, 39, 49 control of, 257–258
fatty acids, 35–39 glycerol phosphate shuttle, 258
lipid composition of membranes, 49 inner membrane, 236–237
lipid–water interactions, 44–47 malate/aspartate shuttle, 258–260
phospholipid monolayers, 47–49 membrane protein complexes
phospholipids, 41–42 ATP synthesis, 247–249
significance, 35 loop mechanism, 245–246
triacylglycerols, 39–41 proton pump, 244–245
water permeability, 49–51 pump and annihilation, 247
Lipid metabolism succinate dehydrogenase, 245
absorption of dietary lipids, 325–326 membrane transport
carbohydrate metabolism, 355–358 adenine nucleotide translocase, 252
cholesterol metabolism, 346–351 phosphate exchange, 252
eicosanoids, 351–352 transport proteins, 252–254
fatty acid biosynthesis, 335–343 superoxide formation by, 256–257
fatty acid oxidation Mixed (noncompetitive) inhibition, 120, 124–125, 456–457
activation, 327–328 Mobile coenzymes, 139
β-oxidation, 330–334 Mobile cofactors, 139
transport, 328–330 Molecular orbitals, 452
in fed and fasted states, 353–355 Monomers, 99
ketone body metabolism, 334–335 Monosaccharides
phospholipid metabolism, 343–346 aldoses and ketoses, 55
sphingolipids, 353 chiral carbon, 56
triacylglycerol formation, 343 chiral molecule, 56
unusual bacterial fatty acids, 353 enantiomers, 56
Lipid-water interface, 457 d-erythrose, 58
Lipoprotein, 49 Fischer projection, 55
Liposomes, 44 d- and l-glyceraldehyde, 56
Logarithms, 449–450 polarimeter, 57
Loops, 93, 97 stereoisomers, 56
Lovastatin, 347 tetrahedral carbon, 56
Lumen, 266 Motif, 94, 96
Lyase enzymes, 132 Multiple dissociable groups, 90
Lysine catabolism, 375 Murphy’s Law, 112
Muscle hypertrophy, 446
M Myoglobin, 98
Macromolecules, 92 N
Major groove, 409
Malate DH, 226–227 NAD binding domain, 97
Malic enzyme, 337 NADPH production, 318
Malin, 322 NDP kinase, 173–174
Maple syrup urine disease, 373 Near-equilibrium, 169
Mass action ratio, 168 Nernst equation, 178
Mass spectroscopy, 107 Neutral phospholipid pathway, 343
Matrix space, 213 N-glycosidic bond, 73
Melanosomes, 10 Nicotinamides, 140–144
Melting temperature, 410 Nitrification, 363
Membrane fluidity, 49 Nitrogenase, 364
Menadione, 140 Nitrogen cycle, 363
Meso form, 77 Nitrogen disposal, 400–402
Mesophyll cells, 282 Nitrogen fixation, 363
Messenger RNA, 417 Nitrogen metabolism
Metabolically irreversible, 169 amino acid metabolism
Metabolic cycle, 211 arginine, proline, and histidine, 377–378
Metal ion cofactors, 154–156 aromatic amino acid biosynthesis, 382–383
Methionine cycle, 380 branched-chain amino acid breakdown, 372
Methotrexate, 397–398 essential amino acids, 382
Methylcellulose, 67 glycine and methionine breakdown, 378–380
Micelles, 44 lysine, 372–374
Michaelis–Menten equation, 115–117 metabolic pathways, 376–377
464 Index
nonessential amino acids, 382 carriers of protons, 237–238

one-carbon (1C) metabolism and serine, 378–380 defined, 235
phenylalanine and tyrosine degradation, 374–376 electrochemical cell and mitochondria, 239–240
threonine, 372 electron pathways
tryptophan, 374 energetics of electron flow, 241–243
metabolically irreversible reaction, 364–365 sequence of electron flow, 240–241
near-equilibrium nitrogen exchange reactions membrane-bound complexes, 238–239
glutamate DH, 366 mitochondria
transaminases, 366–368 control of, 257–258
NH3 assimilation glycerol phosphate shuttle, 258
redox-active reactions, 364 malate/aspartate shuttle, 258–260
redox-balanced reaction, 365 superoxide formation by, 256–257
redox-neutral, 364 mitochondrial inner membrane, 236–237
nitrogen cycle, 363–364 proton-motive force, 250–251
urea cycle uncoupling, 254–256
aspartate in excess, 369 Oxidative stage, 314
citrulline, 369 Oxidoreductases, 132
cytosolic steps, 369–371
in excess [aspartate], 368–369 P
overall urea cycle, 371–372
Non-cooperative, 102 Palmitate synthase, 340
Noncyclic pathway, 274 Parallel beta sheets, 94, 96
Nonpolar amino acids, 83 PARP, 416
Nonreducing end, 66 Partially ionic bond, 17
Nonreducing sugars, 63 Pasteur effect, 190, 229
N-terminus, 91 Path-dependent, 163
Nuclear magnetic resonance (NMR), 107 Path-independent, 163
Nucleic acids Pathway-irreversible, 440
DNA repair, 415–416 Pathway-reversible, 440
histones, 411–412 Pathways, 169
replication, 412–415 Pathway thermodynamics, 201–202
strand structures, 405–406 Pentose phosphate shunt, 209
structure of double helix, 407–410 nonoxidative stage
supercoiling, 410–411 NADPH production and ribose-5-P, 318
transcription, 416–423 pseudo three-dimensional view and Calvin cycle, 316–318
Nucleophilic substitution, 129–131 reactions of, 315
Nucleosides, 70, 72 oxidative stage, 314–
Nucleotide metabolism Peptide bond, 90–92
deoxynucleotide formation, 394–395 Peroxidase, 334
methylation to form dTMP, 395–398 Peroxisomes, 334
nitrogen pathways, 398–399 Phenylalanine, 374–375
purine degradation, 391–393 Pheophytins, 270
purine nucleotide cycle, 394 Phosphatidic acid, 343
purine nucleotide regulation, 393 Phosphoenolpyruvate carboxykinase (PEPCK), 306
purine synthesis, 386–391 Phosphofructokinase (PFK), 190–193
pyrimidine synthesis, 384–386 Phosphofructokinase-2, 192
salvage reactions, 393 Phosphoglycerate kinase, 195
Nucleotides, 72 Phosphoglycerate mutase, 195–196
Nucleus, 11 Phospholipid biosynthesis, 347
Phospholipid dependent kinase (PDK), 302
O Phospholipid metabolism, 343–346
Phospholipid monolayers, 47–49
Odd carbon-numbered fatty acids, 331–334 Phospholipids, 41–42, 454
Odd-numbered fatty acids, 331–334 Phosphorylated sugars, 69
Ohms, 176 Phosphotyrosine kinase, 199
Oils, 40 Phosphotyrosine phosphatase, 199
Okazaki fragments, 413 Photon, 269
Oleaceae, 332 Photosynthesis
Oleate, 332 C3 plants, 282
Oligosaccharides, 64 C4 plants, 282
Organic, 2 Calvin cycle (see Calvin cycle)
Organic chemistry, 2 chloroplasts
Organic farming, 2 CO2 fixation, 268–269
Osmosis, 50 N and P sides of membrane, 266–267
Osmotic pressure, 50 reversed oxidative phosphorylation, 267–268
Outer membrane permeability, 214 cyclic electron transfer and variations, 274
Oxidation model, 7 electron transfer, 270–272
Oxidation number, 8, 175 light absorption and antennae, 270
Oxidative phosphorylation light and carbon reactions, 265–266
carriers of both electrons and protons, 238 light–dark division, 286
carriers of electrons, 237 proton and electron flow, 272–274
Index 465
secondary metabolites, 283 Pyridoxal phosphate, 152–154

sucrose and starch, 284–285 Pyrimidine degradation, 386
Photosynthetic organisms, 265 Pyruvate carboxylase, 149, 305
Photosystem, 270 Pyruvate dehydrogenase complex, 213
Pi (π) bond, 453 Pyruvate kinase, 197–199
Ping-pong, 367 Pyruvate transporter, 213
Plasmodesmata, 282
Plastocyanin (PC), 271 Q
Polar amino acids, 82
Polar bond, 17 Q-cycle, 245
Polarimeter, 57 Quaternary structure, 98–99
Polarimetry, 57
Polarity, 17 R
Polar lipids, 41
Polar molecule, 17 Racemic acid, 77
Polyamines, 411 Racemic mixture, 77
Poly A polymerase, 421 Radical anion, 144
Poly-A tail, 422 Ras protein, 359
Polycistronic, 436 Rate constant, 3
Polymerase chain reaction (PCR), 425–426 Reaction center, 270
Polymers, 64 Reactions
Polyprotic acids, 41 acid–base reactions, 6–7
Polysaccharides, 55 collision theory of reaction rates, 3
branched polysaccharides, 66–68 generic reaction, 2
linear polysaccharides, 66 redox reactions, 7–8
Polyunsaturated fats, 40 Reaction velocity, 113
Porin, 214 Reactive oxygen species, 392
Positive cooperativity, 102, 127 Receptor tyrosine kinases, 303
Postprandial state, 311 Red blood cell, 10
Post-translational modifications, 440 shunt pathway, 203
Prenylation, 359 Redox-active, 101
Pre-steady-state, 6 Redox coenzymes
Pribnow box, 419 flavin coenzymes, 144–146
Primary structure, 93 nicotinamides, 140–144
Primase, 415 ubiquinone, 144
Primitive, 175 Redox reactions, 7–8
Primosome, 414 Reducing end, 66
Probenecid, 392 Reducing equivalents, 258
Processivity, 414 Reducing sugar, 63
Prochirality, 220–221 Reductases, 132
Products, 2 Reductionist, 12
Proline, 377–378 Repetition–variation, 216
Promoters, 418 Replication, 72
Prosthetic group, 100 double helix, 414–415
Proteasome, 442 eukaryotic replication, 415
Protein analysis, 107 initiation, 412–413
Protein degradation primer formation, 414
extracellular proteases, 440–442 and replisome, 413–414
intracellular proteases, 442–443 Replication fork, 413
mTOR pathway, 443–445 Replisome, 413
Protein folding, 99–100 Resistance training, 446
Protein kinase C (PKC), 300 Resonance, 37, 38
Protein purification, 105–106 Resonance energy transfer, 270
Protein synthesis Respiration, 254
co-translational modifications, 437–439 Respiratory distress syndrome, 47
elongation, 433–436 Resting–exercise transition, 312–313
eukaryotic translation, 437 Restriction enzymes, 425
genetic code, 432–433 Reversible enzyme inhibition, 454–457
initiation, 433 Rho protein, 419
post-translational modifications, 440 Ribose-5-P, 318
ribosomes, 431–432 Ribosomal RNA (rRNA), 418
rRNA, 431–432 Ribosome, 431
termination, 436 Ribozymes, 432
tRNA, 429–431 RS System, 221
Protons, 451
P site, 433 S
PTB domain, 96
Purine degradation, 391–393 S6 kinase (S6K), 443
Purine synthesis, 386–391 Saccharide, 55
PV work, 163 S–adenosylmethionine (SAM), 380
Phylloquinone, 140 Saturating, 6
466 Index
Schiff base, 153, 154 enthalpy, 163–164

Secondary metabolism, 212 heat and work, 161–163
Secondary metabolites, 283 free energy, 165–167
Secondary structure nonstandard free energy changes, 168–169
α-helix, 94 origins of, 161
β-sheet, 94 second law of thermodynamics and entropy
Selenium, 156 ratio of heat to temperature, 165
Semialdehyde, 373 statistical distribution of states, 165
Semipermeable, 11, 50 standard free energy, 167–168
Sequential, 367 Thermophiles, 425
Serum albumin, 104 Thiamine pyrophosphate, 151–152
Shine–Dalgarno segment, 433 Thioredoxin reductase, 394
Shuttle pathways, 258 Thrombospondin, 326
Sigma, 452 Thrombospondin receptor, 326
Signal sequence, 440 Thylakoid membrane, 266
Single-stranded binding protein, 414 TIM domain, 96, 97
siRNA, 418 Titration, 30–31
Skeletal muscle and exercise, 446 Topoisomerases, 410
Slow-reacting substances of anaphylaxis, 352 Trans-aldolase, 315
Small G proteins, 359 Transaminases, 153
Soft nucleophile, 84 Transcription, 73
Sp2 molecular orbitals, 452 in E. coli, 419
Spectrophotometer, 113 eukaryotic transcription, 419–423
Sphingolipids, 353 RNA polymerase binding to DNA, 418–419
Splice variants, 199 Trans fats, 40
Src, 97 Transferase enzymes catalyze reactions, 132
Src homology-2 (SH2) binding domains, 96, 301 Transfer RNA (tRNA), 418
Stacking interaction, 408 Transimidization, 153
Standard conditions, 167 Transketolase, 277–279, 315
State variables, 163 Translation, 73
Statins, 156, 346 Translational, 25
Steady-state, 5–6 Translocase, 132
Steady-state assumptions, 115 Translocase enzyme, 133
Stereoisomerism, 76–77 Transporters, 132
Stereoisomers, 56 Triacylglycerol formation, 343
Steroids, 42 Triacylglycerols, 39–41
Sterols, 49 Triose isomerase, 96
Stim1, 97 Triose phosphate isomerase (TIM), 194
Stroma, 266 Type 1 diabetes, 357
Subcutaneous fat, 40 Type 2 diabetes, 357
Substituted carbohydrates, 70–75
Substrate-level phosphorylation, 224 U
Substrates, 2
Succinyl-CoA synthetase, 224 Ubiquinone, 144
Succinyl–CoA transferase, 334 Ubiquitin, 442
Sucralose, 65 Umami receptor, 79
Sucrose, 62, 65 Uncoupling, 252, 254
Sugar acid, 68 Unsaturated fatty acids, 331
Sugar-modified proteins, 75 Unusual bacterial fatty acids, 353
Suicide substrate, 223 Upstream, 418
Supercoils, 410 UQ-cytochrome c reductase, 245
Supercomplexes, 261–262 Ureido, 370
Supersecondary structure, 94, 96 Ureotelic, 400
Surfactant, 47 Uricotelic, 401
Svedberg, 432
Synthase, 132 V
Synthetases, 132
System, 161 Valence electrons, 452
Vasopressin, 92
T Vectors, 449
Vibrational energy, 25
Tartrate crystals, 77 Visceral fat, 40
Telomeres, 415 Vitamin, 140
Temperature, 161 Vitamin B12, 332, 380
Termination codons, 432 Vitamin K, 140, 150–151
Termination sequence, 419 cycle of, 157–158
Tertiary structure, 97–98 Voltage, 176
Thermodynamics, 8 Voltage dependent anion channel (VDAC), 214
first law of thermodynamics
Index 467
W titration and buffering, 30–31

White adipose, 39
Warfarin, 157 Wobble hypothesis, 432
Water Work, 8, 161, 162
acids and bases, 26–27
condensed phase water, 18–21
gas phase water, 15–16 X
Henderson–Hasselbalch equation, 28–30
Xenobiotics, 319
high heat retention, 24–25
X-ray crystallography, 107
hydrophobic effect, 21–23
ionization of, 25–26
molecules soluble in, 23–24 Z
partial charges and electronegativity, 16–18
pH scale, 27–28 Zwitterion, 86

Ochs Biochem

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Ochs Biochem

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Biochemistry

and by CRC Press

© 2022 Raymond S. Ochs

First edition published by Jones and Bartlett Publishers, Inc 2012

CRC Press is an imprint of Taylor & Francis Group, LLC

ISBN: 978-0-367-46553-7 (hbk)

Typeset in Warnock Pro

CONSIDERING METABOLISM ORGANIZATION

A allopurinol – A synthetic drug (a hypoxanthine analog)

of serine residues in glycogen synthase and cytochromes, or at fixed oxidation state, as in

Compared to its component sciences, biochemistry itself is a young discipline. Hoppe-Seyler

1.2 SOME CHEMICAL IDEAS

◾◾ Without specifying a value, what is the meaning of Avogadro’s number?

Box 1.1 Word Origins: Organic

1.2.1 REACTIONS AND THEIR KINETIC DESCRIPTION

FIGURE 1.1 A generic reaction.

FIGURE 1.2 Collision theory of reaction rates.

(1.3) ratef = k f [A][B]

(1.4) rater = k r[C][D]

Box 1.2 Rate Constants

Reaction Order Rate Constant Units

and we can characterize it with the equilibrium constant:

To describe a steady-state, we need a minimum of 3 components, for example, a substrate (S),

Equation (1.11) represents two reactions:

TABLE 1.1 Increasing Incorporation of Oxygen

and the reduction half:

internal energy, enthalpy, and entropy.

1.7 SPECIES HIERARCHY AND EVOLUTION

Evolution is a fundamental principle of biology. It is also commonly misunderstood, perhaps

Box 1.3 Archaea

nucleus spontaneous generation

2.1.1 GAS PHASE WATER

2.1.2 PARTIAL CHARGES AND ELECTRONEGATIVITY

2.1.3 CONDENSED PHASE WATER: HYDROGEN BONDING

Box 2.1 Covalent and Ionic: Discreet or Continuous Distinction?

2.1.4 HYDROGEN BONDING IN CONDENSED PHASE WATER

TABLE 2.3 Boiling

2.2 THE HYDROPHOBIC EFFECT

Box 2.2 Word Origins: Hydrophobic

Box 2.3 A Thermodynamic Look at the Hydrophobic Effect

2.3 MOLECULES SOLUBLE IN WATER

2.4 HIGH HEAT RETENTION: THE UNUSUAL

TABLE 2.4 Solvent Properties

DMF O 153 3.82 47.2 0.04

Water O H 100 1.85 80 36

Formic Acid H 100 1.41 58 5.2

Acetonitrile N 81 3.92 36.6 0.003

2.6 SOME DEFINITIONS FOR THE STUDY OF ACIDS AND BASES

◾◾ In a neutral solution, [H+] = [OH−]

2.8 THE HENDERSON–HASSELBALCH EQUATION

The equilibrium expression for this equation is:

(2.10) K a = éëCH 3COO- ùû éëH + ùû / [CH 3COOH ]

in a similar way to the development of equilibrium constant expressions in Chapter 1. Taking

(2.16) pH = pK a + log éëCH 3COO- ùû / [CH 3COOH ]

Equation (2.16) is the Henderson–Hasselbalch equation, which is a log transformation

(2.17) 7.4 = 4.7 + log éëCH 3COO- ùû / [CH 3COOH ]

(2.21) K a = [ NH 3 ] éëH + ùû / éëNH 4 + ùû

The corresponding form of the Henderson–Hasselbalch equation is:

(2.22) pH = pK a + log [ NH 3 ] / éëNH 4 + ùû

2.9 TITRATION AND BUFFERING

(2.24) 0 = log éëCH 3COO- ùû / [CH 3COOH ]

Taking the antilog of both sides yields:

(2.26) CO2 + H 2O  H 2CO3  H + + HCO3 -

and is maintained by having a relatively high concentration of CO2. Changes in respiration

1.2 SOME CHEMICAL IDEAS

1.2.1 REACTIONS AND THEIR KINETIC DESCRIPTION

1.7 SPECIES HIERARCHY AND EVOLUTION

2.1.1 GAS PHASE WATER

2.1.2 PARTIAL CHARGES AND ELECTRONEGATIVITY

2.1.3 CONDENSED PHASE WATER: HYDROGEN BONDING

2.1.4 HYDROGEN BONDING IN CONDENSED PHASE WATER

2.2 THE HYDROPHOBIC EFFECT

2.3 MOLECULES SOLUBLE IN WATER

2.4 HIGH HEAT RETENTION: THE UNUSUAL

2.6 SOME DEFINITIONS FOR THE STUDY OF ACIDS AND BASES

2.8 THE HENDERSON–HASSELBALCH EQUATION

2.9 TITRATION AND BUFFERING

(2.26) CO2 + H 2O H 2CO3 H + + HCO3 -

3.6 LIPID–WATER INTERACTIONS OF AMPHIPATHIC MOLECULES

3.8 LIPID COMPOSITION OF MEMBRANES

3.9 WATER PERMEABILITY OF MEMBRANES AND OSMOSIS

4.2 RING FORMATION IN SUGARS

5.1 COMMON STRUCTURE OF THE AMINO ACIDS

5.2 BIOLOGY OF THE AMINO ACIDS

5.3 AMINO ACID INDIVIDUALITY: THE R GROUPS

5.4 ACID–BASE PROPERTIES AND CHARGE

5.4.1 TITRATION AND NET CHARGE