How Enzymes Work

“The enzyme is a classic but very important material that everybody in the field of biology,
medicinal chemistry, biotechnology, and medicine must be familiar with. It is the extreme
feature of protein that works in the cells. This concise book covers classic and modern
enzymology and, therefore, is an excellent guide for those who possess the basic knowledge
of chemistry and want to proceed to advanced courses.”
Prof. Takeshi Nishino
University of Tokyo, Japan
How Enzymes Work

“This carefully written book provides very useful information on enzymes and will immensely
benefit not only undergraduate and graduate students but also researchers interested in
enzymes.”
Dr. Hitoshi Nakamoto
Saitama University, Japan
For a long time, enzymes have been studied by measuring their activity, which has
led to the advancement of “enzyme kinetics.” In recent years, the mechanism of
enzyme reaction has been explained in detail on the basis of the 3D structure. Genetic
engineering and the 3D structural analysis of enzymes contribute to these advancements
in enzymology. This book starts with an introduction to various enzymes to show how
interesting enzymes are, which is followed by historical kinetic studies on enzymes and
the overall and rapid-reaction kinetics. The subsequent topics describe the basics of
protein structure, the control of enzyme activity, and the purification of enzymes. A case
on the kinetic and structural studies of l-phenylalanine oxidase is also presented. There
are many good books on enzyme kinetics, but few describe their kinetic and structural
aspects. This book deals with both and contains many references that can be good
sources for further reading. It is handy and is especially helpful for beginners. A number
of figures, including some with stereo expression, facilitate observing the 3D structure
of enzymes.
Haruo Suzuki is professor emeritus at Kitasato University, Tokyo, Japan,

a councilor of the Japanese Biochemical Society, and a member of
the Japan Society for Bioscience Biotechnology and Agrochemistry. A
biochemist, he graduated from the Department of Chemistry, Tokyo
Metropolitan University, in 1966 and received his DSc from the Division of
Suzuki
Biophysics and Biochemistry, the Graduate School of Science, University
of Tokyo, in 1971. He worked as a postdoctoral fellow in the Department
of Pathology, University of California at San Diego, from 1971 to 1973. He worked at
the Institute for Developmental Research, Aichi Prefectural Colony, Japan, from 1973 to
1978, Kitasato University School of Medicine, Japan, from 1978 to 1994, and Kitasato
University School of Science from 1994 to 2007. Prof. Suzuki’s research interests focus on
the computer analysis (QM/MD) of enzyme catalysis.
V424
ISBN 978-981-4463-92-8
How
Enzymes Work
1BO4UBOGPSE4FSJFTPO3FOFXBCMF&OFSHZ7PMVNF
How
Enzymes Work
FROM STRUCTURE TO FUNCTION
editors
Preben Maegaard Haruo Suzuki
Anna Krenz
Wolfgang Palz
The Rise of Modern Wind Energy
Wind Power
for the World
CRC Press
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Contents
Preface xi
1. Introduction 1
1.1 General Properties of Enzyme 1
1.1.1 Enzyme Specificity 2
1.1.2 Rate Enhancement 2
1.2 Examples of Enzyme 4
1.2.1 Neurotransmission and Muscular Action 4
1.2.2 Gastric Juice and Proton Pump 6
1.2.3 Genetic Test of Alcohol Sensitivity and
DNA Polymerase 9
1.2.4 Enzyme Sensor Determination of Glucose 12
2. Overall Reaction Kinetics 17

2.1 Road to the Steady State Kinetics 17
2.1.1 Sucrose Hydrolysis 17
2.1.2 Henri’s Treatment of the Enzymatic
Reaction 19
2.1.3 Michaelis–Menten Equation 20
2.1.4 Briggs and Haldane’s Steady State
Method 24
2.2 Demonstration of the Enzyme–Substrate
Complex 24
2.2.1 Peroxidase Reaction 25
2.2.2 Crystallization of the ES Complex 26
2.3 Meaning of Steady State 27
2.3.1 Steady State Model: Tab Model 27
2.3.2 Application of the Tab Model to the
Enzymatic Reaction 29
vi Contents
2.4 Kinetic Parameters 30

2.4.1 kcat 30
2.4.2 kcat/Km 31
3. Factors That Affect Enzyme Activity 35
3.1 Enzyme Concentration 35
3.2 Substrate Concentration 37
3.2.1 One Substrate Reaction 37
3.2.2 Two-Substrate Reaction 40
3.2.2.1 Ordered bi-bi mechanism 41
3.2.2.2 Random bi-bi mechanism 41
3.2.2.3 Ping-Pong bi-bi mechanism 42
3.3 Inhibitor 43
3.3.1 Reversibility 43
3.3.2 Derivation of Rate Equations 44
3.3.2.1 Competitive inhibition 44
3.3.2.2 Non-competitive Inhibition 45
3.3.2.3 Uncompetitive Inhibition 46
3.3.2.4 Mixed-type inhibition 46
3.3.3 Graphical Method for the Determination
of the Type of Inhibition and Dissociation
Constants 46
4. Effect of pH, Temperature, and High Pressure on

Enzymatic Activity 53
4.1 Effect of pH 53
4.1.1 A Basic Model 53
4.1.2 Graphical Methods to Determine pK Value 55
4.1.3 Meaning of pK Values 58
4.2 Thermodynamics in the Enzymatic Reaction 59
4.2.1 Basics of Thermodynamics 60
4.2.2 Transition State Theory 61
4.2.3 Determination of Thermodynamic
Parameters of the Enzymatic Reaction 64
4.3 Temperature Dependence of the Enzymatic
Reaction 65
Contents vii
4.4 Effect of Pressure 66

4.4.1 Effect of Pressure on the Rate of Reaction 67
4.4.2 Meaning of the Activation Volume 67
4.5 The Effect of Temperature and Pressure on
a-Chymotrypsin-Catalyzed Reaction 68
4.5.1 Ef fect of Temperature 69
4.5.2 Effect of Pressure 71
5. Measurement of Individual Rate Constants 75

5.1 Rapid-Mixing Techniques 75
5.2 Analysis of the First-Order Reaction 79
5.2.1 Order of Reaction 79
5.2.2 Practical Methods to Determine the
First-Order Rate Constant 83
6. Structure of Protein 87
6.1 Amino Acids 87
6.2 Polypeptide and Protein 92
6.3 Analysis of Primary Structure 92
6.3.1 Protein Chemical Methods 93
6.3.2 cDNA Sequencing: Dideoxy Method 96
6.4 Three-Dimensional Structure 99
6.4.1 Weak Interactions 99
6.4.1.1 Electrostatic interaction 99
6.4.1.2 Hydrogen bond 100
6.4.1.3 Hydrophobic interaction 100
6.4.1.4 van der Waals force 101
6.4.2 Secondary Structures and Their Determination 102
6.4.2.1 a helix 103
6.4.2.2 b sheet and b turn 104
6.4.2.3 Determination of secondary
structures 104
6.5 Tertiary and Quaternary Structures 106
6.6 Structural Motif and Loop 108
6.6.1 Supersecondary Structures: Motifs 108
viii Contents
7. Active Site Structure 117

7.1 Active Site and Active Center 117
7.2 Cofactor, Coenzyme, Prosthetic Group 117
7.2.1 NAD+ (Nicotinamide Adenine Dinucleotide)
and NADP+ (Nicotinamide Adenine
Dinucleotide Phosphate) 118
7.2.2 Coenzyme A (CoA, CoA-SH) 119
7.2.3 Flavin Mononucleotide (FMN), Flavin
Adenine Dinucleotide (FAD) 120
7.2.4 Heme 121
7.2.5 Pyridoxal Phosphate (PLP) 121
7.2.6 Folate 123
7.2.7 Thiamine Pyrophosphate 123
7.2.8 Biotin 125
7.2.9 Lipoamide 126
7.2.10 Protein-Derived Cofactors 126
7.3 Search of Active Site 127
7.3.1 Chemical Modification 127
7.3.1.1 Amino group 127
7.3.1.2 Carboxyl group 128
7.3.1.3 Sulfhydryl group 128
7.3.1.4 Hydroxyl group 129
7.3.1.5 Guanidino group 130
7.3.1.6 Imidazole group 131
7.3.1.7 Indole group 131
7.3.2 Site-Directed Mutagenesis 131
7.3.3 Examples of Active Site Studies 132
7.3.3.1 Chemical modification of l-Phe
oxidase 133
7.3.3.2 Site-directed mutagenesis of
thermostable l-lactate
dehydrogenase 136
8. Control of Enzyme Activity 141

8.1 Regulation by Non-Covalent Interaction 141
8.2 Regulation by Covalent Modification 149
Contents ix
8.2.1 Activation of Enzymes by Cleavage of

Polypeptide Chain 149
8.2.2 Regulation by the Side Chain
Phosphorylation 154
8.2.2.1 cAMP-dependent protein kinase,
protein kinaseA (PKA) and glycogen
metabolism 155
8.2.2.2 Regulatory subunit of PKA 156
8.2.2.3 Catalytic subunit and overall
reaction mechanism of catalysis 159
8.2.2.4 Phosphoryl transfer reactions
at the active site of the C subunit 164
9. Preparation of Enzyme 171

9.1 Extraction of Enzyme 171
9.2 Purification of Enzyme 172
9.2.1 Method to Use the Solubility of Proteins 172
9.2.1.1 Salting-out 172
9.2.1.2 Precipitation with organic
solvents 174
9.2.2 Column Chromatography 174
9.2.2.1 Ion exchangers 175
9.2.2.2 Gel filtration 176
9.2.2.3 Affinity chromatography 177
9.3 Purity Analysis of Enzyme 178
9.3.1 Electrophoresis 179
9.3.2 Sodium Dodecyl Sulfate Polyacrylamide
Gel Electrophoresis 179
9.3.3 Isoelectric Focusing 180
10. A Case Study: l-Phenylalanine Oxidase (Deaminating

and Decarboxylating) 185
10.1 Introduction 185
10.2 Preparation of PAO 186
10.2.1 Preparation of the Cell Extracts 186
10.2.2 Purification of PAO by Column
Chromatographies 186
Contents
10.3 Catalytic Properties of PAO 188

10.3.1 Stoichiometry of the Reaction Catalyzed
by PAO 188
10.3.2 Overall Reaction Kinetics 188
10.3.3 Determination of Kinetic Constants 190
10.3.4 Hydrogen Quantum Tunneling in the
PAO-Catalyzed Reaction 192
10.3.4.1 Hydrogen quantum tunneling
(hydrogen tunneling) 192
10.3.4.2 Hydrogen tunneling in the
PAO-catalyzed reaction 195
10.4 Structural Properties of PAO 196
10.4.1 Nucleotide and Its Deduced
Amino Sequences of PAO Gene and
Its Expression 196
10.4.2 3D Structures of proPAO and PAOpt 198
10.5 Substrate Specificity and Reaction
Specificity of PAO 199
Appendix 207
Solutions 211
Index 219
Preface
About three years ago, I received an e-mail from Mr. Stanford

Chong, director and publisher at Pan Stanford Publishing, just after
returning from BIT Life Sciences’ Second Symposium on Enzymes
and Biocatalysis (2011). He proposed that I write a book on
enzymes. After several e-mail exchanges, we agreed to publish this
book.
The prominent nature of enzymes is to enhance the rate of
reaction, and most enzymes are composed of proteins. Therefore,
the first half of the book describes the kinetic aspects of enzymes
following a discussion on the general properties of enzymes
in Chapter 1. Moreover, in Chapter 1, readers will learn how
interesting enzymes are. This part describes the overall reaction
kinetics by introducing historical works. The methods to analyze
the effect of various factors on the enzyme activity and the practical
way to determine the rate constants of the enzymatic reactions
are also presented. The second half of the book describes the
structural aspect of enzymes. Chapters 6 and 7 describe the general
properties of proteins and the method to determine the overall
and the active site structures of proteins. Chapter 8 describes the
control of enzyme activity. It also discusses enzyme regulation by
non-covalent interaction and by covalent modification. Protein
kinase A is discussed in the section on regulation by the side chain
phosphorylation, though the enzyme itself is activated with cAMP.
Chapter 9 describes the standard methods of enzyme preparation.
The functional and structural aspects of a phenylalanine oxidase
(deaminating and decarboxylating) are discussed as a case study
in Chapter 10.
A molecular simulation method that combines the quantum
mechanics (QM) and molecular mechanics (MM) approaches is
commonly used in chemical and biological systems. Chapter 4
shows that enzymatic reactions can be monitored experimentally
at a time scale of milliseconds. A QM/MM approach simulates the
reaction pathway in an enzymatic reaction under the nanosecond
xii Preface
to picosecond level. In Chapter 8, I make a brief mention of the

QM/MM approach. Now, this method is becoming an important
tool in enzymology. The 2013 Nobel Prize in Chemistry was given
to three doctors, Martin Karplus, Michael Levitt, and Arieh Warshel,
for their pioneering works “for the development of multiscale
models for complex chemical systems.”
As much as possible, I have selected original papers as
reference. When it was difficult to get access to old works, I
followed review papers and books. Most of the 3D structures
of proteins are drawn from pdb files by PyMOL software. Some of
the figures are shown by stereo view; therefore, a 3D viewer may
be helpful.
For a better understanding of the text, especially the derivation
of equations, a few problems are presented at the end of
Chapters 2 to 10. The problems are meant to be solved inde-
pendently. To aid the readers in solving the problems, solutions to
difficult problems have been provided at the end of the book.
I thank the following for their comments and advice: Dr. H.
Nakamoto, Saitama University (Chapters 1 to 10), Dr. K. Abe,
Nagoya University (Section 1.2.2 and Box 1.1), Dr. Y. Nishina,
Kumamoto University (Section 4.5.1), Dr. K. Tamura, Kitasato
University (Sections 1.2.3, 6.3.2, and 7.3.2), and Dr. S. Yoneda,
Kitasato University (problem 1 and its solution in Chapter 6 and
Section 8.2.2.4).
Although I have carefully prepared the manuscript, the
possibility of errors cannot be ruled out. Therefore, the readers
are welcome to let me know if they find any error.
I would also like to express my sincere thanks to Mr. Chong
for his kind advice and help, and Mr. Arvind Kanswal, senior
editor, and his editorial team for their patience and kindness
during the publication process.
Haruo Suzuki
Sagamihara, Japan
January 2015
Chapter 1
Introduction
Foods are digested in the human body to glucose, fatty acids,

amino acids, and so on. These are used to produce energy to
work and synthesize biomolecules, such as protein, nucleic acids,
carbohydrate, and lipids. These changes are performed in cells
and called metabolism. The metabolic pathways are composed of
chemical reactions, which are catalyzed by enzyme. This chapter
describes a brief history of early works on enzyme, fundamental
view on the enzyme, and some examples to show its importance.
1.1 General Properties of Enzyme

It seems common that the recognition of enzyme as substance is
derived from the observation of Payan and Persoz in 1833. They
obtained the substance that converted starch to saccharificate
and named it diastase. Then Schwann obtained the substance that
digested meat, and named it pepsin in 1836. Berzelius invented
the term catalysis in 1835 to describe chemical reactions in which
the progress of the reaction is affected by a substance that is not
consumed in the reaction. Diastase was used to mean enzyme,
but later ferment was used to mean microorganism with the
fermentation activity, and also mean the substance like diastase
and pepsin. To avoid the confusion, Kühne proposed the term
enzyme in 1878 to name the substance like diastase. It means in
How Enzymes Work: From Structure to Function

Haruo Suzuki
Copyright © 2015 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4463-92-8 (Hardcover), 978-981-4463-93-5 (eBook)
www.panstanford.com
Introduction
yeast in Greek. Many enzymes have name with suffix—ase, which

originated from Duclaux’s proposal in 1898 [1].
Our life without enzyme could not have been imagined. Our
health is maintained by the proper action of various enzymes, and
we are surrounded by many things containing enzyme, such as
enzyme supplement, detergents, and toothpaste. In this chapter,
first, general properties of enzyme are summarized. Then the
importance of enzyme is shown by some examples.
1.1.1 Enzyme Specificity

The substance on which an enzyme acts is substrate, which is
abbreviated as “S.” Enzyme specificity is classified into substrate
specificity and reaction specificity. In other words, an enzyme has
“liking” for substrate on which it acts and for the reaction that it
catalyzes.
Substrate specificity is that an enzyme acts on its restricted
substrate. However, enzymes show a different degree of specificity.
For example, alcohol dehydrogenase catalyzes dehydrogenation
of ethanol with high efficiency, but alcohol dehydrogenase catalyzes
dehydrogenation of methanol with low efficiency. Such an enzyme
is considered to have “low or broad substrate specificity.” Urease
only catalyzes the hydrolysis of urea to produce ammonia and
carbon dioxide; thus, it is called to have high or narrow substrate
specificity.
In reaction specificity, an enzyme catalyzes a particular
transformation of the substrate. For example, l-amino acid oxidase
catalyzes the oxidation of l-amino acid to produce the corresponding
keto acid, ammonia, and hydrogen peroxide. However, the
racemization of l-amino acid to d-amino acid is catalyzed by the
enzyme different from l-amino acid oxidase, that is, amino acid
racemase. See Chapter 10 for the example of the reaction specificity.
1.1.2 Rate Enhancement

The prominent nature of the enzyme is the enhancement of a
reaction rate. The enhancement is calculated by dividing the
enzyme-catalyzed rate by the uncatalyzed rate, and is in the range
of 106–1016 times of the rate of uncatalyzed reaction (Table 1.1) [2].
When substrate changes to product, substrate must pass over an
General Properties of Enzyme
energy barrier (Fig. 1.1). The energy is called activationdenergy,

ln k E.
= a2
Arrhenius (1889) introduced a relationship between dT a rateRT
of
reaction (k) and temperature (T ), the Arrhenius equation:
d ln k E
= a2 (1.1)
dT RT
where
d ln k E and R are the activation energy and the gas constant
= a 2 –1
(8.314RT
dT J K ), respectively. This equation explains our experience
that the reaction rate increases with increasing temperature.
Figure 1.1 Energy profiles of uncatalyzed and catalyzed reactions.

Substrate (S) passes over the energy barrier (activation
energy) to be transformed to product (P) in the presence and
absence of enzyme (E). Energy barrier is lower with enzyme.
Table 1.1 Rate enhancement by enzymes
Enzymes Rate enhancement

Adenosine deaminase 2.1 × 1012
Alkaline phosphatase 1.6 × 1016
Chorismate mutase 1.9 × 106
Peptidase 1.5 × 1010
Triose phosphate isomerase 6.2 × 109
Urease 2.3 × 1013
Abzyme (amide hydrolysis) 2.5 × 105
Source: Adapted from Suzuki [2].
Introduction
Determination of the activation energy: Integrating Eq. 1.1,
 –E 
k = A exp  a  (1.2)
 RT 
where A is pre-exponential factor. Logarithm of Eq. 1.2 results in
–Ea
ln k = + ln A (1.3)
RT
Thus, the ln k vs. 1/T plot will give a linear line (Fig. 1.2). The
activation energy of a reaction could be estimated from a slope of
the plot. A most popular theory to explain the kinetics of reaction
is the transition state theory. When a reaction proceeds, a substrate
(ground state) passes over the unstable transition state (Fig. 1.1).
The energy required passing over the barrier is called activation
energy. The enzyme reduces the activation energy and thus
increases the rate of reaction. See Chapter 4 for detail.
Figure 1.2 Arrhenius plot. From the slope of the plot, the activation
energy can be determined.
1.2 Examples of Enzyme

1.2.1 Neurotransmission and Muscular Action
Muscular action is controlled by the motor neuron. When the brain
asks to move a hand, the signal reaches the muscular cells through
Examples of Enzyme
the neuronal transmission. In this transmission, the signal from one

neuron to another neuron is performed by the neurotransmitter,
acetylcholine. The reason why the transmitter is required is that
these neurons are not directly connected. The junction is called
“synapse.” At the synapse, one neuron (presynaptic cell) releases
acetylcholine, which is diffused to the other neuron or the target
cell like muscular cell (postsynaptic cell) (Fig. 1.3).
Figure 1.3 Neurotransmission Action potential reaches prosynaptic

cell  Na+ enters  Membrane potential changes  Potential-
dependent Ca2+ channel opens  Ca2+ enters  Exocytosis
of synaptic vesicle (SV)  Release of acetylcholine (ACh) 
ACh binds receptor  Generation of action potential. ACh
esterase (AChE) hydrolyzes ACh to produce choline (Ch) and
acetate. Ch is transported back to presynaptic cell by choline
transporter (CHT) and used to form ACh.
Thus, the signal transmits presynaptic to postsynaptic cells.

After the signal transmission, acetylcholine should be removed.
If not, the signal is continually transmitting. To avoid this, the cell
hydrolyzes acetylcholine by acetylcholine esterase. The enzyme
catalyzes the following reaction:
Introduction
CH3COOCH2CH2N+(CH3)3 + H2O  CH3COO– + H+ + HOCH2CH2N+ (CH3)3
To perform the rapid transmission of signal, the esterase has

quite high catalytic activity (around 104 s–1); thus, the acetylcholine
must be hydrolyzed in several tens of microseconds [3, 4]. Without
this enzyme, acetylcholine stays at the receptor and muscle is
keeping contracted. The site of enzyme that is important for the
enzyme activity is called active site; which will be considered in
Chapter 7. Ser residue is in the active site. The highly poisonous
sarin irreversibly reacts with this Ser residue and thus inactivates
the enzyme. The reaction is called chemical modification, which will
be described in Chapter 7. Sarin’s poison has been known by the
sarin gas attack on the Tokyo Subway incident in 1995.
1.2.2 Gastric Juice and Proton Pump

Our foods are sent to the stomach, mixed with gastric juice, and
digested. The gastric juice is highly acidic, about pH 1 (0.1 M H+).
Let’s discuss how our body is able to produce acidic conditions
under mostly neutral conditions of our body (around pH 7.4)
(Fig. 1.4). The enzyme concerned is “proton pump.” In addition to
the pump, carbonic anhydrase is also involved. This enzyme catalyzes
the formation of H+ and HCO–3 from CO2 and H2O [5, 6]:
CO2 + H2O  H+ + HCO–3 (1.4)
The catalytic activity of carbonic anhydrase is one of the

highest of all enzymes [6]. The turnover rate ranges from 104 to
107 s–1. This means that one enzyme catalyzes the above reaction
104 to 107 times per second.
The active site is located inside molecule, where a zinc ion
is coordinated with three nitrogen atoms of His residues. The
zinc ion constitutes the active site and is involved in the catalytic
mechanism. The carbonic anhydrases are widely distributed in
living systems, and have various functions, such as homeostasis
of acid–base balance, transport of CO2 and HCO–3 , and supply of H+
and HCO–3 . The gastrointestinal carbonic anhydrase supplies H+
as gastric juice and serves HCO–3 to neutralize the acidic digests in
the duodenum.
Examples of Enzyme
Figure 1.4 The mechanism of formation of gastric juice. HK, H+, K+-
ATPase; CA, carbonic anhydrase; KCC4, K+, Cl– cotransporter;
Mt, mitochondria.
The proton pump, H+, K+-ATPase, carries H+ through the

membrane of the gastric cell against about 106-fold H+ concentration
gradient. The process that carries ions and substances against
their concentration gradient is called active transport. This process
is similar to “pump” and therefore is called proton pump. The
pump needs energy, and the gastric proton pump uses the energy
formed by the ATP hydrolysis.
The H+, K+-ATPase belongs to the P-type ATPase, such as
Na , K+-ATPase and Ca2+-ATPase, and is bound to the parietal cell
+
membrane. The reaction mechanism of H+, K+-ATPase is shown

schematically in Fig. 1.5 [7]. The enzyme has two conformations, E1
and E2. The ion-binding site of the E1 form faces the cytoplasmic side,
and binds with two protons and ATP to form the phosphorylated
form {E1P(H+)2}. The carboxy group of Asp385 is the site of
phosphorylation. Morii et al. proposed that two protons are
transported via charge transfer pathway and a water wire from the
cytoplasm to the inside of the enzyme protein [8]. The ion-binding
Introduction
site of the E1P(H+)2 form is faced to lumen, and the E1P(H+)2 form
changes its conformation to the E2P form to release 2H+. The E2P
form binds with 2K+, which binding induces the dephosphorylation
of the E2P form to release Pi. Finally ATP and 2H+ bind to the
E2(K+)2 form with concomitant release of 2K+ to the cytoplasm to
form E1(H+)2ATP. As four steps are reversible processes, the step,
E1P(H+)2 to E2P, must be reversed under high H+ concentration
of lumen. If this is the case, the gastric H+ gradients must not be
formed. Abe et al. cleared the problem by the structural studies on
the pig H+, K+-ATPase by the Electron Crystallography [7, 9]. The
enzyme is a heterodimer protein composed of α and β subunits.
The α subunit contains 3 domains and 10 transmembrane α
helix, and 3 domains are in the cytoplasmic side. The β subunit is
composed of one transmembrane α helix. The N-terminal tail of
β subunit is interacting with the phosphorylation domain of α
subunit to stabilize the E2P form, thus preventing the reverse
reaction of E2P to E1P(H+)2. As for the stoichiometry of the mole
proton transported per mole ATP hydrolyzed, two protons can
be released in exchange for 2K+ without violating the amount
of energy available from ATP hydrolysis at pH > 3, but only a
single proton can be transported per ATP hydrolyzed at pH < 3
[7, 8, 10].
Figure 1.5 Action mechanism of H+, K+-ATPase. Notice that the proton
release to lumen is irreversible. Adapted with permission from
Abe, K., Seikagaku, 84, 115–119, 2012 [7].
Examples of Enzyme
Box 1.1
Electron Crystallography and Cryoelectron Microscopy
The X-ray crystallography uses an X-ray beam to illuminate sample,
and electron microscope (EM) uses an electron beam while light
microscope uses visible light. The difference between light and
electron microscopes is in the wavelength difference. The wavelength
of electrons in EM is about 105 times shorter than visible light.
This means, theoretically, the resolution of EM to be of atomic
levels. However, severe radiation damage caused by electron beam
irradiation has limited structural analysis of biological specimen.
Prof. Fujiyoshi and colleagues contributed greatly to the development
of the method to observe the biological samples by EM. They invented
EM to overcome various problems incurred upon applying EM to
the biological materials. One problem was the damage to samples
caused by the illumination of electron beams, and the second was
the vaporization of water in samples under vacuum in the EM. They
invented EM, which is able to be used under Cryo temperature (very low
temperature, such as liquid N2 or liquid He temperatures) to lower the
damage of samples (Cryo-electron microscopy), and they also invented
the method of sample preparation to prevent the vaporization. When
we use the two-dimensionally-crystalline samples (2D crystals)
such as sheet or helical tubular crystals, electron diffraction and/or
Fourier transformation of their micrographs allow us to determine
three-dimensional structure of biological macromolecules (Electron
crystallography). The ability to obtain structural information from 2D-
ordered arrays makes this approach particularly useful for studies of
membrane proteins in liquid bilayer. In addition to the H+,K+-ATPase
structure, structures of bacteriorhodopsin, water channel aquaporin
1, and acetylcholine receptor were reported.
Reference: Fujiyoshi, Y. and Unwin, N. (2006) Electron crystallography
of proteins in membranes. Curr. Opin. Struct. Biol., 18, 587–592.
1.2.3 Genetic Test of Alcohol Sensitivity and DNA

Polymerase
Orientals have higher alcohol sensitivity (facial flushing, elevation
of skin temperature, increase in pulse rate) than Caucasians. This
is mostly derived from the activity of aldehyde dehydrogenase.
10 Introduction
Ingested alcohol (ethyl alcohol) is oxidized by alcohol dehydrogenase

to produce toxic acetaldehyde, which is then oxidized by aldehyde
dehydrogenase to non-toxic acetate.
C2H5OH  CH3CHO  CH3COO– (1.5)
Humans have more than 12 aldehyde dehydrogenase genes

(ALDH), but the ALDH2 gene is mostly related to the alcohol
sensitivity [11]. Aldehyde dehydrogenase 2 (ALDH2) is a tetrameric
enzyme composed of four identical subunits. Each subunit
contains about 500 amino acid residues. The alcohol sensitivity is
associated with the deficiency of the enzyme activity due to the
mutation of the residue 487Lys to Glu, a point mutation of a G to A
transition.
Glu (GAA)  Lys (AAA) (1.6)
The mutation occurs in the exon 12 of the ALDH gene. There are
three combinations of normal ALDH2 (N) and mutant ALDH2 allele
(M): NN, NM, and MM. People with NN gene possess high ALD activity
(normal activity), people with NM gene possess about 10% of the
normal ALD activity, and people with MM gene possess almost 0%
of the normal ALD activity. People who possess NM or MM gene are
highly sensitive to alcohol.
Genetic testing of the ALDH2 gene can be performed by
polymerase chain reaction (PCR) and the agarose gel electrophoresis
of the PCR products. Mullis invented the PCR method [12, 13] and
was awarded the Nobel Prize in Chemistry in 1993 for the invention.
Polymerase chain reaction is the method to amplify the region
of DNA interested. Figure 1.6 shows how the PCR method works.
First, we must design primers with about 20 nucleotides in length,
one is complementary to the 3¢-region of anti-sense sequence
(sense primer), and the other is complementary to the 3¢-region
of sense sequence (antisense primer). Template DNA, sense and
antisense primers, and dNTPs are mixed with thermostable DNA
polymerase, and are heated to about 90°C, leading to separate DNA
strands. Then the whole mixture was cooled to about 60°C, leading
to bind each primer with the corresponding sequence of DNA
chains. By heating to about 70°C, DNA polymerase catalyzes the
synthesis of complementary DNA, starting from each primer.
Thus, DNA chain is amplified twice by one cycle of these reactions.
Examples of Enzyme 11
Therefore, if we perform 20 cycles of PCR, we will obtain 106-fold

of the amplified DNA.
Figure 1.6 Amplification of dsDNA by polymerase chain reaction.
In the case of aldehyde dehydrogenase (ALDH2), the exon 12 of

ALDH2 gene is amplified using the sense primer, and the normal (N)
and mutant (M) antisense primers [11, 14]. The sequences of the
primers are
Sense primer: 5-CAAATTACAGGGTCAACTGCT-3
Normal antisense primer: 5-CCACACACTCACAGTTTTCTCTTC-3
Mutant antisense primer: 5-CCACACACTCACAGTTTTCTCTTT-3
The underlined triplets correspond to antisense sequences of
Glu and Lys residues. The size of the amplified region is 135 base
pairs. Figure 1.7 shows the pattern of the agarose gel electrophoresis
of the amplified DNA. It is clearly shown that the NN homozygote
was amplified only by the normal (N) primers, the NM heterozygote
was amplified by the normal and mutant primers, and the MM
homozygote was amplified only by mutant (M) primers. Like this, we
could determine one’s ALDH2 gene-type from a few pieces of hair.
12 Introduction
Figure 1.7 Agarose gel electrophoresis pattern of the PCR-amplified

DNA samples. The normal ALDH2 (NN homozygote), normal-
mutant ALDH2 (NM heterozygote), and mutant ALDH2 (MM
homozygote) samples. N, normal; M, mutant.
1.2.4 Enzyme Sensor Determination of Glucose

Determination of glucose is used in a clinical laboratory for the
diagnosis of diabetes. A simple method is to use a paper containing
glucose oxidase, peroxidase, and chromogen. When one dips the
paper in glucose-containing urine, the white paper changes to
yellow. Thus, we can see whether or not urine contains glucose. The
principle of the determination is as follows. The glucose oxidase
catalyzes the reaction,
Glucose + O2  Glucono-δ-lactone + H2O2. (1.7)
H2O2 produced oxidizes the colorless chromogen to the colored

chromophore by the action of peroxidase.
Chromogen + H2O2  Chromophore + H2O (1.8)
Familiar chromogens are ABTS {2,2¢-azino-bis(3-

ethylbenzothiazoline-6-sulfonic acid), changes green by oxidation}
and TMB (3,3¢,5,5¢-tetramethylbenzidin, changes blue by oxidation).
This method is easy, and one may get the test paper at the
drug store. However, the method is rather qualitative. For the
quantitative determination, the glucose sensor was first developed
Examples of Enzyme 13
by Clark [16]. The sensor is composed of the Clark-type oxygen

electrode covered with the oxygen-permeable membrane, which
is then covered with the membrane immobilized with glucose
oxidase (Fig. 1.8). When the electrode is dipped in the glucose-
containing solution, the following reactions take place.
On the glucose membrane: Glucose consumes oxygen to form
H2O2. At cathode (platinum), oxygen is reduced:
O2 + 4H+ + 4 e–  2H2O (1.9)
At anode (silver), solid silver is oxidized:
4Ag + 4Cl–  4AgCl + 4e– (1.10)
By this electrode, one can easily determine the glucose content as

the oxygen consumed. Figure 1.8 shows an electrode for glucose
sensor [15, 16]. If you have an oxygen electrode and recorder, you
can easily construct a glucose sensor. The amount of oxygen
consumed may be hindered by the contaminating reducing
substances. To avoid this, the method to detect the hydrogen
peroxide using the hydrogen peroxide electrode had been invented
[15, 16].
Figure 1.8 Electrode for glucose sensor. GO, polyethylene membrane

with immobilized glucose oxidase. SM, semipermeable
membrane. The membranes are tightly fixed by O-ring to the
oxygen electrode. KCl, KCl solution. Anode and cathode are
connected to recorder.
14 Introduction
References
1. Dixon, M., and Webb, E. C. (1958). Enzymes. Longmans, Green and Co.
London, New York, Toronto.
2. Suzuki, H. (1994). Recent advances in abzyme studies. J. Biochem., 115,
623–628 (review).
3. Quinn, D. M. (1987). Acetylcholinesterase: Enzyme structure, reaction
dynamics, and virtual transition states. Chem. Rev., 87, 955–979.
4. Silman, I., and Sussman, J. L. (2008). Acetylcholinesterase: How
is structure related to function? Chem. Biol. Interact., 175, 3–10
(Review).
5. Lindskog, S. (1997). Structure and mechanism of carbonic anhydrase.
Pharmacol. Ther., 74, l–20.
6. Hilvo, M., Baranauskiene, L., Salzano, A. M., Scaloni, A., Matulis, D.,
Innocenti, A., Scozzafava, A., Monti, S. M., Di Fiore, A., De Simone, G.,
Lindfors, M., Jänis, J., Valjakka, J., Pastorekova, S., Pastorek, J.,
Kulomaa, M. S., Nordlund, H. R., Supuran, C. T., and Seppo Parkkila, S.
(2008). Biochemical characterization of CAIX, one of the most active
carbonic anhydrase isozymes. J. Biol. Chem., 283, 27799–27809.
7. Abe, K. (2012). Unique properties of gastric H+,K+-ATPase and
conserved conformational changes among P-type ATPases.
Seikagaku, 84, 115–119. (mini-review in Japanese).
8. Morii, M., Yamauchi, M., Ichikawa, T., Fujii, T., Takahashi, Y., Asano,
S., Takeguchi, N., and Sakai, H. (2008). Involvement of the H3O+-Lys-
164–Gln-161–Glu-345 charge transfer pathway in proton transport
of gastric H+,K+-ATPase. J. Biol. Chem., 283, 16876–16884.
9. Abe, K., Tani, K., Nishizawa, T., and Fujiyoshi, Y. (2009). Inter-subunit
interaction of gastric H+,K+-ATPase prevents reverse reaction of the
transport cycles. EMBO J., 28, 1637–1643.
10. Abe, K., Tani, K., Friedrich, T., and Fujiyoshi, Y. (2012). Cryo-EM
structure of gastric H+,K+-ATPase with a single occupied cation-
binding site. Proc. Natl. Acad. Sci. U S A., 109, 18401–18406.
11. Yoshida, A., Rzhetsky, A., Hsu, L. C., and Chang, C. (1998). Human
aldehyde dehydrogenase gene family. Eur. J. Biochem., 251, 549–557.
12. Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G. T., Erlich, H.
A., and Arnheim, N. (1985). Enzymatic amplification of beta-globin
genomic sequences and restriction site analysis for diagnosis of
sickle cell anemia. Science, 230, 1350–1354.
References 15
13. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T.,
Mullis, K. B., and Erlich, H. A. (1988). Primer-directed enzymatic
amplification of DNA with a thermostable DNA polymerase. Science,
239, 487–491.
14. Braun, T., Bober, E., Singh, S., Agarwal, D. P., and Goedde, H. W. (1987).
Isolation and sequence analysis of a full length cDNA clone coding for
human mitochondrial aldehyde dehydrogenase. Nucl. Acids Res., 15,
3179.
15. Cass, A. E. G. (1990). Biosensors. A Practical Approach. The Practical
Approach Series (IRL Press at Oxford University Press).
16. Clark, Jr., L. C., and Lyons, C. (1962). Electrode systems for continuous
monitoring in cardiovascular surgery. Ann. N. Y. Acad. Sci., 102,
29–45.
Chapter 2
Overall Reaction Kinetics
This chapter describes the early history of enzyme kinetics.

Enzyme kinetics is the study of the chemical reactions that are
catalyzed by enzyme. The goal of enzyme kinetics is to describe
the catalytic mechanism of enzyme on the basis of kinetic data.
By learning the history, you may understand the basis of enzyme
kinetics.
2.1 Road to the Steady State Kinetics

Biochemistry books describe the Michaelis–Menten mechanism of
the enzyme-catalyzed reaction. However, there were many works
before the establishment of the mechanism [1, 2].
2.1.1 Sucrose Hydrolysis

The inversion of sucrose to glucose and fructose was easily
monitored by a polarimeter. Sucrose is dextrorotatory (specific
rotation: [a]25
D = 66.5°). Glucose is dextrorotatory (α-d-glucose;
[a]25
D
= 112.2°), and fructose (β-d-fructose, [a]25
D = –132°) is
levorotatory, and the whole products become levorotatory after
mutarotation of each product. Thus, sucrose is inverted to the
levorotatory products.

Haruo Suzuki
www.panstanford.com
18 Overall Reaction Kinetics
These facts lead to the products called invert(ed) sugar. Here,

mutarotation is the change in the optical rotation that occurs by
epimerization. In this case, the direct product of sucrose is α-d-
glucose and β-d-fructose. For example, α-d-glucose is gradually
converted to β-d-glucose, becoming in equilibrium between two
epimers. Two epimers show different specific rotation, then the
optical rotation changes. Mutarotation ends immediately by the
addition of the alkaline solution.
Wilhelmy studied the acid hydrolysis of sucrose quantitatively
in 1850. The rate of hydrolysis of sucrose is expressed by Eq. 2.1:
dx
= k( a – x ) (2.1)
dt
where a and x represent the initial concentration of sucrose and
the sucrose hydrolyzed at time t, respectively. By integrating
Eq. 2.1 and by introducing the initial conditions, t = 0, and x = 0,
a
kt = ln (2.2)
a– x
Applying Eq. 2.2 to their data, the first-order rate constant was
determined. Extending the acid-catalyzed hydrolysis of sucrose,
O’Sullivan and Tompson (1890) studied the invertase-catalyzed
hydrolysis of sucrose [3]. They stopped the reaction by adding
alkaline solution to the reaction mixture, allowing mutarotation to
completion. They found that the catalytic activity is proportional to
the concentration of invertase, and that sucrose prevents the heat
denaturation of invertase. These suggest the complex formation of
invertase with sucrose. Wurts (1880) had found that papain forms
insoluble compound with fibrin. Fischer (1894) proposed a key
and lock hypothesis to explain the specificity of glycosidases. These
observations led to the concept of enzyme–substrate complex in
the current form.
Road to the Steady State Kinetics 19
2.1.2 Henri’s Treatment of the Enzymatic Reaction

The quantitative analysis of the enzymatic reaction was first
reported by Henri (1903). He studied the hydrolysis of sucrose by
invertase, the hydrolysis of salicin by emulsin, and the hydrolysis of
starch and dextrin by amylase. These works revealed that (1) the
rate of hydrolysis linearly increases with increasing concentration
of substrate in the low substrate concentrations, and becomes
constant at the high concentrations, and (2) the rate increases
linearly with increasing concentrations of invertase and is inhibited
by the addition of the product.
Henri proposed two schemes to explain these results [1, 2]:
Scheme 1: E + S 

 ES (2.3)
k 
E + S   E + P (2.4)

Scheme 2: E + S 
 ES
k
ES   E + P (2.5)
In schemes 1 and 2, the enzyme reversibly forms the complex with

substrate, ES, but the complex is not able to form the product in
scheme 1. On the other hand, the complex changes to the product
in scheme 2. In scheme 1, the product is formed by a bimolecular
reaction. Moreover, it was assumed that the enzyme forms the
complex with its product:

E + P 
 EP (2.6)
Applying the mass action law to Eqs. 2.3 and 2.6, we get
[ES] [EP] (2.7)

[E][S] = m [E][P] = n
The total concentration of enzyme ( e0)=is[E] + [ES] + [EP]
e0 = [E] + [ES] + [EP] (2.8)

A substance in brackets is defined to be a molar concentration of

the substance. Introducing a and x to the initial concentration of
substrate and the concentration of the product formed at time t,
respectively,
e
[E]= 1 + m(a –0 x ) + nx

me (a – x )
[ES]= 1 + m(0a – x ) + nx

Then the rate of the enzymatic reaction can be expressed by the

following equations:
dx ke0(a – x )
In scheme 1, v = = k[E][S]= (2.9)
dt 1 + m(a – x ) + nx

dx kme0(a – x )
In scheme 2 v = = k[ES]= (2.10)
dt 1 + m(a – x ) + nx

Equations 2.9 and 2.10 have the different coefficient but are
homomorphic. Then these equations can be expressed as Eq. 2.11
dx a
when the initial velocity (v0 )=was C 1 + ma
dt =measured:
dx a
v0 = dt = C 1 + ma (2.11)

where C is the constant. This equation is the same form as the well-
known Michaelis–Menten equation.
The above results are very important. That is, the ES complex
is the dead-end and abortive complex in scheme 1, but the
compulsory or obligatory complex in scheme 2. However, the rate
equation is essentially the same form. This means that, only by
measuring the rate of overall reaction, one could not determine
which scheme is correct. To clarify this, kinetic and structural
studies as described in Chapters 5 and 6 are required.
2.1.3 Michaelis–Menten Equation

We now know that the enzyme activity is affected by the H+
concentration in the reaction media. Sörenson (1909) pointed
the importance of H+ concentration for the enzyme activity, and

introduced the concept of pH [4]:
pH = –log [H+]
Henri’s work was performed before Sörenson’s report, so the effect

of [H+] was not considered. Moreover, Henri studied the invertase
reaction without considering the mutarotation. Michaelis and
Menten (1913) analyzed the invertase reaction under the conditions
to prevent these problems [5]. They performed the experiments
under the constant pH using acetate buffer, and the reaction
was stopped by the addition of alkaline solution to complete the
mutarotation. In addition to these, they introduced the initial rate
measurement (Fig. 2.1). Determining the initial rate, one can remove
the secondary effects such as denaturation of enzyme, change of
pH, and accumulation of product. Michaelis and Menten analyzed
the rate data by applying Henri’s scheme 2. That is, they assumed
that the enzyme forms the complex with substrate by a rapid
equilibrium (or quasi-equilibrium):
+1 k k+2

E+S   ES   E+ P (2.12)
k
–1
The initial rate (v) is expressed as follows:
k e s _______
v = _______ = Vs
+2 0
(2.13)
K s + s Ks + s
V = k+2e0 (2.14)
[E][S]
k–1 _______
Ks = ____ =
(2.15)
k+1 [ES]
where e0, s, V, and Ks, are the total concentration of enzyme, the
initial concentration of substrate, the maximum rate, and the
dissociation constant of ES complex, respectively.
To determine V and Ks, Michaelis and Menten determined v at
various concentration of substrate, and plotted against log s.
Figure 2.1 An initial rate measurement. By measuring the initial rate

(line 1), we can determine accurately the rate of reaction
without considering the secondary effects on the enzyme
(lines 2 and 3). At the times shown by •, the rate is smaller
for lines 2 and 3 than line 1.
From Eq. 2.13,
__ s
v = _______ (2.16)
V Ks + s
From Eq. 2.16, a plot between v/V and log s will show a sigmoidal
curve as shown in Fig. 2.2. At the inflection point, log s = log Ks,
and the slope of the curve,
d(v /V ) 2.303
v= = = 0.576
d(log s ) 4
Using the property, they plotted v vs. log s, and normalized the
scale of v to give the slope of 0.576, where v/V = 0.5. Thus, V and
Ks were obtained. The dissociation constant Ks obtained was
0.0167 M. The reciprocal of Ks is the association constant; this
means that they determined the affinity of the enzyme with
substrate for the first time.
Figure 2.2 Hydrolysis reaction of sucrose by invertase. The relation

between the initial rate (v; relative value) and the concentration
of sucrose (s). log Ks = –1.78 was obtained at v/V = 0.5. Adapted
from Michaelis, L., and Menten, M. L. (1913) Biochem. Z., 49,
333–369.
Van Slyke and Cullen (1914) analyzed the urease-catalyzed

reaction by assuming that ES does not reform E and S,
k+1 k+2
E+S   ES   E+ P (2.17)
and obtained the rate Eq. 2.18 similar to the Michaelis–Menten

equation [6]. 1Briefly,
11 the1 times required for the first and second
 =  =+ +
steps are 1/k+1s and
kk+1+2s1/k+2, respectively: then the time required to
complete one cycle is
1 1
= +
k+1 s k+2
k e :s
Then, v can be expressed for the enzyme concentration
v = +2 0
k+2
+s
k es k+1
v = +2 0
k+2 (2.18)
+s
k+1
2.1.4 Briggs and Haldane’s Steady State Method

As described in the above section, Michaelis–Menten and Van
Slyke-Cullen introduced the rate equation under the conditions,
d[ES]
d[ES] d[ES]
d[ES]
dt dt +1 +1 dt –1dtkk–1+1
= k =[E][S]–
k [E][S]–
k [ES]–
= =»[E][S]–
[ES]–
kk+1+2[E][S]–
[ES]=
kand
+2[ES]=
k–10
[ES]– « k+2[ES]=
k–10 [ES]– , krespectively.
+2[ES]=
0 0 These are the extremes of
conditions. Briggs and Haldane (1925) introduced more general
rate equation [7]. They assumed that the time-dependent change
of ES concentration is zero during the reaction. For the reaction
(Eq. 2.12), Eqs. 2.19 and 2.20 are generated:
d[ES] (2.19)
dt = k+1[E][S]– k–1[ES]– k+2[ES]= 0
d[P] k+2e0 s (2.20)

v= = k+2[ES]=
dt (k–1 + k+2 )
+s
k+1
In Eq. 2.20, the Michaelis–Menten equation (2.13) is generated

d[ES]
d[ES]
= k = [E][S]–
k [E][S]–
when k–1[ES]– » k+2[ES]=
k–1 [ES]– ,kand
+2[ES]=
0the0 Van Slyke–Cullen equation (2.18) when
d[ES] dt dt
d[ES] +1 +1
= k =[E][S]–
dt dt +1 +1k [E][S]–
k –1 [ES]–
k –1 «
[ES]–
k [ES]=
. k
+2 +2 [ES]=
Thus, 0 0
the Briggs-Haldane method (steady state method)
is more generalized than the previous methods.
The first term in the denominator of Eq. 2.20 is usually expressed
as follows:
k–1 + k+2
Km = (2.21)
k+1

As Michaelis and Menten established the basis of the kinetic
treatment of the enzymatic reaction, and introduced the rate
equation. Therefore, in memory of their work, Eq. 2.20 is called the
Michaelis–Menten equation, and the parameter in Eq. 2.21 is named
as the Michaelis constant. The suffix was initially “M” [8], but “m”
is now preferably being used.
2.2 Demonstration of the Enzyme–Substrate

Complex
The ES complex was assumed to explain the rate of enzymatic
reaction as shown in the previous section. However, in those days,
there was no direct evidence to prove the existence of ES complex
in the enzymatic reaction. The following examples may be the
good ones that demonstrated the existence of ES complex.
Demonstration of the Enzyme–Substrate Complex 25
2.2.1 Peroxidase Reaction

The ES complex has a short life as an intermediate in the enzymatic
reaction. Therefore, a special method was required to detect the
ES complex. The stopped-flow method is usually used to analyze
a rapid reaction. The principle of the method is shown in Fig. 2.3.
Two syringes are connected as shown, and one syringe contains
a solution of enzyme (E) and the other does that of substrate (S).
Push the head of each syringe at the same time, the enzyme solution
is mixed with the substrate solution at the mixing point (M). Then
the reaction after mixing can be monitored by the detector. Various
kinds of detectors are invented, such as those of absorbance,
fluorescence, and circular dichroism (CD). The usual stopped-
flow apparatus is equipped with the detectors of absorbance and
fluorescence. Chapter 6 describes more in details the stopped-flow
method.
Figure 2.3 Principle of stopped-flow method.
Peroxidase contains heme as the prosthetic group, catalyzes

the oxidation of a substance in the presence of H2O2, and shows a
characteristic spectrum around 400 nm. It has been known that the
spectrum changes by the addition of H2O2, indicating the formation
of the E · H2O2 complex (the ES complex in the Michaelis Menten
mechanism). Chance (1943) utilized this nature of peroxidase,
and showed an ES complex as the intermediate in the peroxidase-
catalyzed reaction using the stopped-flow apparatus [9]. Peroxidase

(E) catalyzes the oxidation of a leuco-pigment (AH2) in the presence
of hydrogen peroxide:
k
+1

E + H2O2   E . H2O2
k–1
k
E . H2O2 + AH2 
+2
 E+ A +2H2O
The left-hand syringe contained peroxidase, and the right-hand

syringe contained H2O2 and leuco-malachite green, an equal volume
of both solutions was mixed by pushing the head of syringe. Then
the absorbance change at 400 nm was continually monitored to
detect ES complex, and at 610 nm to detect the malachite green
(A). The absorbance (ES complex) at 400 nm rapidly increased
and reached plateau, then gradually decreased. On the other hand,
the absorbance (A, product) at 610 nm increased after a short
period of lag phase (see Fig. 2.4 for “lag”). Applying the kinetic
constants determined independently to the above mechanism, the
concentration changes of the ES complex and of product agreed
well with the calculated changes of the ES complex and of product.
Thus, the existence of the ES complex was demonstrated as the
reaction intermediate in the enzymatic reaction after 30–40 years
since the reports by Henri, Michaelis, and Menten.
2.2.2 Crystallization of the ES Complex

It might be the first case for the p-hydroxybenzoate hydroxylase (E)
by Yano et al. [10] that the existence of ES complex was demonstrated
as its crystals. The enzyme contains FAD as a cofactor (see
Chapter 7). The enzyme catalyzes the following reaction, and the
FAD cofactor changes from and to the oxidized (Eox)+ and S 
reduced
 EoxS
EoxS + NADPH + H+  + NADP+
(EredS) forms.
p-hydroxybenzoate (S) + NADPH + O2 + H+

 protocatechuate (P) + NADP+ + H2O
The catalytic mechanism is explained by the following reactions:
Eox + S 
 EoxS
Meaning of Steady State 27
EoxS + NADPH + H+ 
 EredS + NADP+
EredS + O2 
 Eox + P + H2O
The mechanism suggests that the enzyme–substrate complex (EoxS)+ NADPH + H+   EredS
Eoxdoes + H+ 
not convert
S + NADPH to NADP+ added NADPH. They utilized this
 EredS +without
+
property of the enzyme and crystallized the EoxS +complex.
NADPH + HThe  EredS + NADP+

solution of the crystal was stoichiometrically reduced by
the concomitant addition of NADPH solution and produced
protocatechuate by the addition of oxygen. The findings clearly
showed that the crystal obtained corresponds to the catalytic
intermediate, and demonstrates, as a crystalline form, the presence
of the ES complex proposed nearly 50 years ago.
2.3 Meaning of Steady State

Briggs and Haldane proposed the general treatment to introduce
the rate equation of the enzymatic reaction. They assumed steady
state, that is, the concentration of the ES complex does not change
with time. Here we consider the steady state.
2.3.1 Steady State Model: Tab Model

Let us imagine a brook pool. The stream of water pours in and out
the pool constantly, but the surface level of the pool is constant.
Thus, the level of the surface of the pool is in the steady state. We
experienced this kind of phenomena, such as the intravenous drip
in a hospital. These might be simply modeled as shown in Fig. 2.4.
When water is poured in the empty tab at a mL/min and out
at kx mL/min, the surface level of water in the tab goes up gradually
and becomes constant (x mL) after a certain time. The time-
dependent change of x can be expressed as follows:
dx
= a – kx (2.22)
dt
The differential equation can be solved, and we get Eq. 2.23 by
introducing the initial condition (at time 0, x = 0).
a
x  (1– e – kt ) (2.23)
k
Using this equation, the time-dependent change of the water

level (x) of the tab is simulated. The amount of water x increases
exponentially, and reaches constant when t » 1/k. Thus, dx/dt =
d(a/k)/dt = 0. This means steady state.
The rate of water accumulation in the reservoir is,
dp
= kx (2.24)
dt
After substituting “x” in Eq. 2.23 into 2.24, the integration of the
derived equation under the conditions (p = 0 at time 0, and p = p at
time t) yields
a (2.25)
p  at – (1– e – kt )
k
In the steady state,
a (2.26)
p  at –
k
Thus, the lag period (t in Fig. 2.4) is determined by introducing
p = 0 to Eq. 2.26:
1
t t 
k
(a) (b)
Figure 2.4 Tab model of steady state. (a) Water flow via pool (tab).
(b) In (a), the time-dependent change of x is simulated
assuming a = 50 mL/min and k = 2 min–1. At 2.3 min, x is
99% of the steady state level, and at 4.5 min, x becomes the
steady state level. p is the time-dependent accumulation of
water from the tab, and increases linearly after the lag period
(~0.5 min). See text in detail.
Meaning of Steady State 29
2.3.2 Application of the Tab Model to the Enzymatic

Reaction
How the Tab model can be applied to the Michaelis Menten-type
enzymatic reaction (Eq. 2.12). Assuming the concentrations of
enzyme, substrate, ES complex, and product as e, s, x, and p,
respectively, and the initial concentrations of enzyme and substrate
dx
dx
kk+1
 +1((e
as e00 ––and
xx)(
)(ss00,––respectively,
xx ––pp)–(
)–(kk–1–1 ++Eq.
kk+2
+2)2.27
)xx is deduced:
dt
dt
dx
 k+1 (e0 – x )( s0 – x – p)–(k–1 + k+2 )x (2.27)
dt
dx dx
 k+1Usually, sk –»(xe–;–pthen
(e0 – x )( )–(
x )( sk0–1–+x k–+2p))–(
x k–1 + k+2 )x
dt dt 0+1 0
dx
= k+1(e0 – x )s0 –(k–1 + k+2 )x (2.28)
dt
= k+1e0 s0 –( k+1 s0 + k–1 + k+2 )x
Comparing Eq. 2.28 with Eq. 2.22,
a = k+1e0 s0
k = k+1 s0 + k–1 + k+2 (2.29)

Thus, a/k is constant, and dx/dt = d(a/k)/dt is 0.

When we study the enzymatic reaction, we measure the rate
of overall reaction, and determine the kinetic parameters. In these
cases, we usually apply the steady state method without knowing
whether the rates are determined under the steady state conditions.
Let us estimate the time required to achieve the steady state of
the enzymatic reaction. As shown in Fig. 2.4b, it might be concluded
that the reactions attained practically “steady state” when “x”
3.0
becomes 95–99% of the steady level. Here, the time ( t 0.95)<required
k
to reach the 95% level of the steady state is estimated. In Eq.+2 2.23,
x = 0.95 × (a/k),
a a
0.95× = (1– e – kt0.95 )
k k
Then,
e – kt0.95 = 0.05
Calculating logarithm of both sides and applying Eq. 2.29,
3.0 3.0
t 0.95   (2.30)
k k+1 s0 + k–1 + k+2
In the denominator of the equation, k+2 is equal to the turnover

number of the enzyme and is usually determined by dividing
dx
the maximum rate with the total concentration of enzyme,  k+1 (e0.– x )( s0 – x – p)–(k–1 + k+
dt
However, other kinetic constants are not always determined
easily. Then the following equation can be generated from Eq. 2.30:
3.0
t 0.95 <
k+2
When an enzyme activity is assayed by using spectrophotometer,

we add the enzyme solution to the reaction mixture containing
substrate, and mix well, then start to measure the absorbance
change of the solution. The time required to start its measuring may
be 10 s at longest. Most of the enzyme has the turnover number
3.0
higher than 1 s–1, meaning that t 0.95 <
is shorter than 3 s, and more than
k
95% of enzyme must be in the steady+2state after 3 s. Therefore, the
rate of the enzymatic reaction has been usually determined under
the steady state conditions.
2.4 Kinetic Parameters

2.4.1 kcat
dx
The catalytic constant,
k e [S] k = k+2, can be calculated by  kV/
+1 (e0 –using
x )( s0 – x – p)–(k–1 + k+2 )x
v = cat 0 = cat–1[E][S] dt
Eq. 2.14. The
k unit
eK[S]of k isKsm , meaning how many catalytic cycles one
+ [S]
v = cat 0 m = cat [E][S]
molecule ofKenzyme
m + [S] turns
K m
over per second. Thus, it is called turnover
number. It is worth to mention that one molecule of enzyme has
not always only one catalytic site, but more than one catalytic site.
When one molecule of enzyme has “n” independent catalytic sites,
dx
kcat e0[S] kcat is calculated to be k+1 (e0.– x )( s0 – x – p)–(k–1 + k+2 )x
 V/n
v= = [E][S] dt
K m + [S] K m
Kinetic Parameters 31
2.4.2 kcat/Km
According to the Michaelis–Menten mechanism of the enzymatic
reaction, the rate (v) of the reaction is expressed under [S] « Km:
kcat e0[S] kcat

v= = [E][S] (2.31)
K m + [S] K m
since most of k enzyme

e [S] kare not bound to the substrate.
v = cat 0 = cat [E][S]
As kcat/K m +is[S]equal
kcat e0[S] K m to {k+1 k+2/(k–1 + k+2)}, the value has the
v= = [E][S]
dimension
K m + [S] K m of the rate constant of the second order reaction
between free enzyme and free substrate and reaches k+1 when
k–1 « k+2. The value indicates the reactivity of enzyme with
substrate, thus it is called specificity constant [11]. Consider this
point further.
Suppose that an enzyme catalyzes the transformation of two
competing substrates, S1 and S2, to the corresponding products,
P1 and P2, respectively, and that the catalysis follows the
Michaelis–Menten kinetics.

E + S1  ES1 
  E + P1 (2.32)


E + S2  ES2 
  E + P2 (2.33)

Then, the rates of the reactions are
k 
v1 = cat  [E][S1 ] (2.34)
 K m 1

k 
v2 = cat  [E][S2] (2.35)
 K m 2

The ratio v1/v2 is
v1 (kcat /K m )1[S1 ]
=
v2 (kcat /K m )2[S2 ]
This equation kmeans e [S]that

k the enzyme specificity is determined
by thekcat e0[S]of kcat/K m +values
ratio [S] Kofm one substrate to the other [11].
v= = [E][S]
K m + [S] K m
k e [S] k
k e [S]The k /K m +value
[S] of
K menzyme is sometimes used as the efficiency
Kofm +enzyme,
[S] K m since the value is proportional to the frequency of
association of enzyme with the substrate (see Eq. 2.31). The upper
k e [S] and
limit of the value is diffusion-controlled k in the range of 108
to 10 M s [12]. The
10 –1 –1
[S] k /K m +values
k e high [S] Kare
m observed with, for
example, acetylcholine esterase
K m + [S] K m(1.6 × 10 8
M s
–1 –1
), triosephosphate
isomerase (2.4 × 10 M s ), and superoxide dismutase
8 –1 –1
(7 × 109 M–1 s–1). These values are in the range of the diffusion-limited

rate, so these enzymes are called “highly developed enzymes”.
Problems
(1) Concerning a plot in Fig. 2.2, introduce and confirm that the
slope at the inflection point is 0.576.
(2) Derive Eq. 2.23.
(3) In Fig. 2.4b, the tag period was shown to be 0.5 min. Deduce
this value.
(4) In Fig. 2.4b, calculate the level of water in the tab (% of the
steady state level) after 2 min of the initiation of the water
flow.
References
1. Segal, H. L. (1959). The development of enzyme kinetics, in The

enzymes (Boyer P.D., Lardy, H., and Myrbäck, K., eds.) 2nd ed., pp. 1–48,
Academic Press, New York.
2. Nakamura, T. (1993). Enzyme Kinetics Japan Scientific Societies
Press (in Japanese).
3. O'Sullivan, C., and Tompson, F. W. (1890). LX.-Invertase: A contribution
to the history of an enzyme or unorganised ferment. J. Chem. Soc.
Trans., 57, 834–931 (review).
4. Sörensen, S. P. L. (1909). Enzymstudien. II: Mitteilung. Über die
Messung und die Bedeutung der Wasserstoffionenkonzentration
bei enzymatischen Prozessen. Biochem. Z., 21, 131–304.
5. Michaelis, L., and Menten, M. L. (1913). Die Kinetik der invertinwirkung.
Biochem. Z., 49, 333–369.
6. Van Slyke, D. D., and Cullen, G. E. (1914). The mode of action of
urease. J. Biol. Chem., 19, 141–180.
References 33
7. Briggs, G. E., and Haldane, J. B. S. (1925). L. Note on the kinetics on

enzyme action. Biochem. J., 19, 338–339.
8. Hofstee, B. H. J. (1952). On the evaluation of the constants Vm and KM
in enzyme reactions. Science, 116, 329–331.
9. Chance, B. (1943). The kinetics of the enzyme–substrate compounds
of peroxidase. J. Biol. Chem., 151, 553–579.
10. Yano, K., Higashi, N., and Arima, K. (1969). p-Hydroxybenzoate
hydroxylase: Conformational changes in crystals of holoenzyme vs
holoenzyme–substrate complex. Biochem. Biophys. Res. Commun., 34,
1–7.
11. Fersht, A. (1999). In Structure and Mechanism in Protein Science,
pp. 116–117, W. H. Freeman and Co., New York.
12. Stroppolo, M. E., Falconi, M., Caccuri, A. M., and Desideri, A. (2001).
Superefficient enzymes CMLS, Cell. Mol. Life Sci., 58, 1451–1460
(review).
Chapter 3
Factors That Affect Enzyme Activity
The previous chapters described briefly that the enzyme activity is

affected by the concentration of substrate and of product. This and
the following chapters describe the effect of factors affecting the
enzyme activity more in detail. What can we get by studying the
effect of various factors on the activity of enzyme? These studies
will lead to a construction of the reaction mechanism, and will give
us the thermodynamic parameters of the reaction.
3.1 Enzyme Concentration

In Chapter 2, we have known that the enzymatic reaction is
expressed by the Michaelis–Menten mechanism:
k+1 k
+2

E + S 
 ES   E + P (3.1)
k–1
Under the steady state of reaction, the rate of the reaction is
expressed by the Michaelis–Menten equation:
k+2 e0 s
v (3.2)
Km + s
k–1 + k+2
Km 
k+1

Haruo Suzuki
www.panstanford.com
36 Factors That Affect Enzyme Activity
Thus, as Eq. 3.2 shows, plotting v vs. the enzyme concentration

gives a linear relation (line A in Fig. 3.1) when the concentration
of substrate is sufficiently larger than that of the concentration of
enzyme.
Figure 3.1 The effect of the concentration of enzyme on the enzymatic

activity.
Sometimes, the data like lines B and C will be obtained. In these

cases, the Michaelis–Menten kinetics could not be applied. At least
two cases are conceivable when these data were obtained. One is
artifacts caused by the experimental conditions, and the other
is the nature of the enzyme itself. In the first case, the enzyme
preparation and/or the reaction mixture may contain an inhibitor
or an activator of enzyme. In the case of line B, the enzyme
preparation may contain an activator of enzyme, but the activator
may dissociate at the low concentration of the enzyme. Or the
reaction mixture may contain an inhibitor of enzyme, thus inhibiting
at low concentration of enzyme. However, high concentrations
of enzyme consume the inhibitor and prevent the inhibition.
Similarly, in the case of line C, the enzyme preparation may contain
the inhibitor of enzyme, thus the inhibitor dissociates from the
enzyme at the low concentration of enzyme, showing relatively high
activity. Or the reaction mixture may contain the activator of the
enzyme, leading line C. In these cases, removal of the inhibitor or
activator in the reaction mixture including enzyme itself may cause
Substrate Concentration 37
the linearity in the plot of the activity vs. enzyme concentration.

Even though an inhibitor or an activator was removed, the enzyme
still gives the data like lines B or C. This means that the enzyme has
its own nature to show such a result. These considerations lead
the important conclusion that the kinetic experiment must be
performed using the highly purified enzyme.
3.2 Substrate Concentration

3.2.1 One Substrate Reaction
When the rate of the enzymatic reaction is expressed by Eq. 3.2,
the v vs. [S] plot is shown in Fig. 3.2.
Figure 3.2 The effect of the concentration of substrate on the enzymatic

activity. Figure is drawn for the hypothetical enzyme having
V = 10, and K m = 2. Error bar: upward, simple error of 5%
of V; downward, relative error of 10% of v. Each error bar
should be both sides, but only one side is shown for simplicity.
Under the constant enzyme concentration, k ev sincreases with

v = +2 0
increasing concentrations of substrate. When s « K m ,
k+2e0 s (3.3)
v=
Km
and the rate linearly increases with the increase of the substrate
concentration.k+2e0 s
v=
When s » K m , the rate becomes independent of the substrate
concentration:
v = k+2 e0 = V
k es
v = +2 0
kV e s
and K m are the important parameters of enzyme, since V and
v = +2 0
K m determine the efficiency of enzyme and kan eapparent
s affinity of
v = +2 0
enzyme with substrate, respectively. Strictly K m is not equal to K s,
because K s = k–1/k+1. However, the step of ES to P usually includes
the transformation of covalent bond. Therefore, the substrate
binding and release with enzyme must be rapidly achieved k e s as
v = +2 0
compared with the step of ES to P. Thus the assumption of K m = K s
k es
is reasonable as the first approximation.
v = +2 0
Determination of V and K m is rather hard practically from the
saturation curve (Fig. 3.2). For example, in the case of the Michaelis–
Menten type enzymatic reaction, the ksubstrate
es concentration to
v = +2 0
give the rate of 0.1V is calculated to be kK+2me/9
0 s from Eq. 3.2, and the
v=
substrate concentration to give 0.9V is 9 K m . Therefore, to increase
the rate from 0.1V to 0.9V (9 times enlargement), the concentration
of substrate must be increased to 81 times of the concentration
of substrate to give the rate of 0.1V. This indicates that, by these
large changes of the substrate concentration, we only get the rate
of 0.9V. This is derived from the fact that the v vs. [S] line kis+2ea 0 s
v=
part of an equilateral hyperbola with the asymptotes of [S] = – K m
and v = V (see Fig. 3.2). To obtain the rate of reaction, the rate of
formation of product or the decrease of substrate is measured.
It is usually assumed that the error in the static measurements is
negligible as compared with the dynamic measurements. In
Fig. 3.2, the error in the substrate concentration is assumed to be
negligible, but the error in the rate measurements is not. Here, it is
worth mentioning our own static error in the measurements. For
example, when we prepare the substrate solution, pipette is usually
used. The handling of pipette is rather simple work. However,
it is recommended to check our own handling of pipette before
experiments. For example, take 10 μL of water by a pipette, and
measure its weight. Repeat these 10 times. Then we will find our
precision in our handling of the pipette. In Figs. 3.2 and 3.3, error
bars are shown. A simple error is that the error region is constant
irrespective of the magnitude of v value. A relative error is that the
error region is dependent on the magnitude of v. That is, the error
region is larger with the larger v [1].
(a) (b)
(c)
Figure 3.3 Linear plots for the hypothetical enzyme in Fig. 3.2. (a) 1/v vs.
1/[S]. (b) [S]/v vs. [S], and (c) v vs. v/[S]. Error bar: upward,
simple error of 5% of V; downward, relative error of 10%
of v. In C, leftward bar, simple error of 5% of V, and rightward
bar, relative error of 10% of v. Each error bar should be
both sides, but only one side is shown for simplicity.
To overcome this problem caused from the v vs. [S] plot for
the determination of the kinetic parameters, several linear plots
were derived from the Michaelis–Menten equation [2–4]. Three

representative plots are as follows:
1 1 Km 1
(A)  +
v V V [S]
(B) [S] = Km + [S]

v V V
v
(C) v = V – K m
[S]
These are shown in Fig. 3.3 and have been named such as
Lineweaver–Burk plot (A), Hanes plot (B), and Eadie plot (C). However,
these namings are not correct historically k e[5];
s therefore, they are
v = +2 0
not used hereafter. From the plot, V and K m are easily determined.
However, the problem in using the plot of 1/v vs. 1/[S] is that
v value with relatively large error at low concentration of substrate
(high value of 1/[S]) affects the slope of the plot, leading to the error
in the determination of the kinetic parameters. Although the plot
has these problems, the 1/v vs. 1/[S] plot is widely used. Among
the three plots, the [S]/v vs. [S] plot is recommended. The statistical
method to estimate the kinetic parameters is written by Cornish-
Bowden [1].
3.2.2 Two-Substrate Reaction

In Chapter 2, the Michaelis–Menten equation was introduced. The
reactions considered were the hydrolytic reactions, so strictly saying,
these are two-substrate reactions. However, the concentration of
water (1000/18 = 55.6 M) is much higher than that of the substrate
(usually mM level); so the change in the water concentration is
negligible, and the step with water is not considered. There are
many reactions with two and more substrates. Here, the
representative mechanism of two-substrate reactions is considered.
The mechanism is named using the number of substrate and
product, and enzyme reactions are graphically expressed by the
method of Cleland [6]. The enzyme is represented by a horizontal
line, and substrate additions and product releases are represented
by vertical arrows. The nomenclature by Cleland uses A, B, and P, Q

as the substrate and product, respectively; these are not used here.
3.2.2.1 Ordered bi-bi mechanism

Two substrates change to two products. The substrate (S1 and S2)
bindings and product (P1 and P2) releases are ordered.
In the scheme, the horizontal line indicates the state of enzyme,

and the arrow shows the forward direction of binding and release
of ligands, but the rate constants of both forward and backward
directions are shown. (ES1S2—EP1P2 ) indicates the interconversion
between substrates and products in the active site of enzyme. The
kinetic behavior of horse liver alcohol dehydrogenase showed the
ordered bi-bi mechanism [7].
3.2.2.2 Random bi-bi mechanism
Two substrates change to two products, but the order of

the substrate binding and the products release is random.
The rate constants are not shown in the mechanism below.
The good example of the mechanism has been reported for
phosphomevalonate kinase [8]. The ordered bi-bi and random bi-bi
mechanisms are called sequential mechanism, since the products
are formed only after binding two substrates with enzyme. The
next “Ping-Pong mechanism” is different from these mechanisms.
3.2.2.3 Ping-Pong bi-bi mechanism

Enzyme (E) binds with substrate (S1) to produce the product
(P1 ) and changes to the F form of enzyme. The F form binds with
S2 to produce P2 and changes to the original form of enzyme, E.
The oxidoreductase and pyridoxal phospohate-dependent

enzymes usually belong to this mechanism. The kinetic study on
glucose oxidase is a good example of the Ping-Pong bi-bi mechanism
[9].
The rate equation of these mechanisms can be drawn by the
initial rate measurements, then [P1 ] = 0 and [P2 ] = 0 can be applied.
Equation 3.4 can be used for the ordered bi-bi and random bi-bi
mechanisms, and Eq. 3.5 for the Ping-Pong bi-bi mechanism. In
the case of the random bi-bi mechanism, Eq. 3.4 is derived by
assuming the rapid equilibrium and the step of ES1S2 to EP1P2 to
be rate-limiting.
1 1 K  1 1 1
= 1+ 2 +  K 1 + K 2K 3  (3.4)
v V  [S2 ]  V  [S2 ] [S1 ]
1 1 K  K 1
= 1+ 2 + 1 (3.5)
v V  [S2 ]  V [S1 ]
1 1 K 11 K 2  1 1  1
1 1 1
      = 1+ 2= +1+ K 1 +  K+2K 3 K1 +K 2K 3 
K 1 1 11 V: Kthe maximum
where 11 v1 V  Kvelocityv12 ] 
2[S 1V V
1obtained
+[SK2 ]K V1 [S2 ]1[S1 ], [S2 ] [S
with 1 ]∞;
1+ 2 +  K 1 + K 2K 3 = 1+ 2 + = K +
1+K K  +  K 
[S2 ]  V  K
v
[S :
] V
[S ] to
[S give
] vVV/2V 1 2 3
under
[S ][S 1 V
] 
[S
1 1
∞;
] K
2 3
: [S ]
1 to
[S give
] V/2 1 under
1
1 1 K  121 1 2 1 1  2  2  1
=  1+ 2 2  1
 K 1 + K 2K 3
+complex. 
= 1+ 2 + [S1]K +∞.K K : dissociation
 constant of the ES
v V  [S2 ] 1 V  [S2 ] [S1 ]
v V  [S2 ]  V  1 2 3
[S2 ] [S1 ]
The difference between the sequential and Ping-Pong
mechanisms is clear as shown in Fig. 3.4. Then, how can we
discriminate between the ordered and random bi-bi mechanisms?
These mechanisms are distinguishable by their initial-rate behavior
toward inhibitors [10].
Inhibitor 43
(a) (b)
Figure 3.4 Reciprocal plots of bi-bi mechanisms. [S2]1 < [S2]2 < [S2]3.
(a) Sequential mechanism. (b) Ping-Pong bi-bi mechanism.
3.3 Inhibitor
Substances that inhibit the enzyme-catalyzed reaction are called
the inhibitor and that enhance the reaction rate are called the
activator. Studies on the effects of these substances reveal the
action mechanism of the enzyme, and will help to develop novel
drugs to cure patients. Here, a simple reversible inhibition will be
described. Irreversible inhibition will be described in Chapter 7.
3.3.1 Reversibility
For the study of a reversible inhibition, first, the reversibility of
inhibition should be tested. Figure 3.5 shows a simple way to test
the reversibility. At first, the effect of concentration of inhibitor
on the activity of enzyme must be determined (line a in Fig. 3.5).
Then the enzyme was incubated with various concentrations of
inhibitor for a given time (for example, 5 min), and a portion of the
mixture was transferred into the assay mixture containing substrate
to determine the enzyme activity. This resulted in diluting the
concentrations of inhibitor. Figure 3.5 shows the enzyme activities
obtained by 10 (b) and 50 times (c) dilution, assuming that the
inhibitor reversibly binds with the enzyme. When an inhibitor binds
with enzyme irreversibly, the enzyme will show a lower activity
than that observed by the reversible binding with inhibitor. As for

the reversible inhibition, competitive, noncompetitive, uncompetitive,
and mixed-type inhibitions are described below.
Figure 3.5 Reversibility test of inhibition. An inhibitor bound with a

hypothetical enzyme reversibly, and showed a competitive
type of inhibition. Ki = 20 μM, Km = 100 μM, and the enzyme
activity was determined with 200 μM substrate. (a) The enzyme
activity was determined in the assay mixture containing the
inhibitor concentrations shown. (b,c) After incubation of
enzyme with the inhibitor, a portion of the mixture was added
to the assay mixture and the activity was assayed. Thus, the
inhibitor concentration was diluted 10 (b) and 50 (c) times.
3.3.2 Derivation of Rate Equations

3.3.2.1 Competitive inhibition
This type of inhibition is induced by the binding of inhibitor (I) to
the binding-site of substrate in an enzyme; thus, the inhibitor
competes with substrate to bind the same site, and produces an
inactive complex EI.
k +1 k

E+S 
 ES 
+2
 E+ P
k –1
Ki

 EI
E+ I 
 (3.6)
Inhibitor 45
The rate of reaction (v) is introduced by applying the steady

state, and expressed assuming that the total enzyme concentration
kis+2e0:
v=
1+(1+[I]/ K i )(K +2 /[S])
k+2e0
v= (3.7)
1+(1+[I]/ K i )(K +2 /[S])
As V = k+2e0,
v=
1+(1+[I]/ K i )(K +2 /[S])
V
v= (3.8)
1 +(1 +[I]/ K i )(K m /[S])

3.3.2.2 Non-competitive Inhibition

This type of inhibition is caused by the binding of the inhibitor with
the enzyme and the enzyme–substrate complex to produce inactive
complexes, EI and ESI. The substrate can bind with the EI complex.
k+1 k

E + S  +2
 ES 
k 
 E+ P
–1
Ki
 EI
E+ I 
K
i

ES + I  ESI (3.9)

K
S
EI +S  ESI
 (3.10)
In this case, the rate equation is introduced by the “rapid equilibrium”

assumption. That is, the equilibria in the above scheme are rapidly
reached before the step ES  E + P occurs. The binding of inhibitor
and substrate with enzyme is independent each other. Then, the
dissociation constants of complexes are assumed to be as shown
in Eqs. 3.1, 3.6, 3.9, and 3.10, then the rate of reaction (v) is
V (3.11)
v=
(1+[I]/K i ) (1+ K s/[S])
V
v=
where
(1+[I]/K means the dissociation constant of the ES complex in
i ) (1+ K s/[S])
Eq. 3.1.
3.3.2.3 Uncompetitive Inhibition

This type of inhibition is caused by the binding of the inhibitor with
the ES complex.
k +1 +2 k

E+S 
 ES   E+ P
k –1
k+3

ES + I 
 ESI
k–3
The rate of reaction is introduced by applying the steady-state

method.
V
v= (3.12)
1+[I]/K i + K m/[S]

where Ki is the dissociation constant of ESI.
3.3.2.4 Mixed-type inhibition

The reaction scheme is similar to non-competitive inhibition.
k +1 k

E+S 

+2
 ES   E+ P
k –1
Ki

EI
E + I 

K s¢ K¢

 i
 ESI ES + I 
EI +S   ESI

Notice that the dissociation constants of the ESI complex are different
from those in the non-competitive inhibition (Eqs. 3.9 and 3.10).
Applying the rapid equilibrium assumption, the rate of reaction is
V
v= (3.13)
1 + [I]/K ¢i + (1+ [I]/ K i )(K s /[S])

3.3.3 Graphical Method for the Determination of the

Type of Inhibition and Dissociation Constants
For the four types of inhibition, the following equations are
derived [11, 12]:
Inhibitor 47
Competitive inhibition:
1 1 K m  [I]  1
= + 1 +  (3.14)
v V V  K i [S]
Non-competitive inhibition:
1 1 [I]  Ks  [I]  1 .
= 1+ + 1+  (3.15)
v V  K i  V  K i [S]
Uncompetitive inhibition:
1 1 [I]  K 1
= 1+ + m (3.16)
v V K i  V [S]
Mixed-type inhibition:
1 1 [I]  K s  [I]  1
= 1+  + 1+  (3.17)
v V
 K ¢i  V  K i [S]

The reciprocal plot between 1/v and 1/[S] clearly discriminates the
1 1of
type  inhibition
[I]  K s (Figs. 1
[I]3.6a–3.9a). 1 [I]  K s of the
1 determination
For the [I]  1
=  1+  + 1+  = 
1+  + 1+ 
dissociation
v V  K i constant,
¢
 V  K i, the[S] following Equations
v V  are
K i derived;
¢ V  K i [S]
can be determined, using Eqs. 3.18–3.22, from the plots shown in
Figs. 3.6b–3.9b,c which were known as the Dixon plot [13].
Competitive inhibition:
1 1 K m  K m
= 1+ + [I] (3.18)
v V  [S]  VK i [S]
Non-competitive inhibition:
1 1 K s  1 K s  1 (3.19)
 1+ + 1+  [I]
v V  [S]  V  [S] K i
Uncompetitive inhibition:
[S] 1 [S]
= ([S]+ K m )+ [I] (3.20)
v V VK i
(a) (b)
Figure 3.6 Competitive inhibition. (a) Reciprocal plot, 1/v vs. 1/[S]. At
the constant concentration of inhibitor, v was determined at
various concentrations of substrate. The plot gives a linear
line. The slope of the line increases with increase of the
inhibitor concentration, but the lines cross at the same point
of the ordinate. (b) 1/v vs. [I] plot. The linear line obtained
with [S]1 crosses with the line obtained with [S]2. The [I]
value at the intersection is equal to –Ki.
(a) (b)
Figure 3.7 Non-competitive inhibition. (a) Reciprocal plot, 1/v vs.

1/[S]. At the constant concentration of inhibitor, v was
determined at various concentrations of substrate. The
plot gives a linear line. The slope of the line increases with
the increase of the inhibitor concentration, and the lines
intersect at the same point on the abscissa. (b) The 1/v vs. [I]
plot. The linear lines obtained with [S]1 and [S2] cross at the
same point on the abscissa. The [I] value at the intersection
is –Ki.
Inhibitor 49
(a) (b)
Figure 3.8 Uncompetitive inhibition. (a) Reciprocal plot, 1/v vs. 1/[S].
The plot gives a linear line. The slope of the line is constant
at various concentrations of inhibitor, producing parallel
lines. (b) The [S]/v vs. [I] plot gives a linear line with the
constant [S]. The line obtained with [S]1 crosses with that
obtained with [S]2. The [I] value at the intersection is –Ki.
(a) (b)
(c)
Figure 3.9 Mixed type inhibition. (a) Reciprocal plot, 1/v vs. 1/[S]. The
plot gives a linear line. The slope of the line increases with the
increase of the inhibitor concentration, and the lines intersect
on one point. The 1/v value of the point is higher than 0 when
Ki < K i¢ (the case shown in this figure), and minus when Ki >
K i¢ . (b) The 1/v vs. [I] plot gives a linear line with the constant
[S]. The line obtained with [S]1 crosses with that obtained with
[S]2. The [I] value at the intersection is –Ki. (c) Vap is obtained
as in (a), and V/Vap is plotted against [I].
Mixed-type inhibition:
1 1 K s  1 1 K  (3.21)
 1+ +  + s  [I]
v V  [S]  V  K ¢i K i [S] 


V [I]
= 1+ (3.22)
Vap K ¢i
where Vap is the rate obtained at [S]  ∞.
Box 3.1
Methanol Poisoning and Liquor
Alcohols are digested in liver by the action of alcohol dehydrogenase

(ADH). Human ADH family members have been categorized into
five classes. The enzyme family has allelic variations among racial
populations. Alleles ADH1B*1 and ADH1B*2 are predominant among
Caucasians and East Asians, respectively. Ethanol and methanol
are converted to aldehydes by the action of ADH and aldehyde
dehydrogenase (ALDH):
ADH ALDH
C2H5OH  → CH3CHO  → CH3COOH
CH3OH → HCHO → HCOOH

Methanol poisoning is derived from the products by the action of

ADH and ALDH. The formaldehyde and formic acid are responsible
for the metabolic acidosis and the loss of sight (optic nerve damage).
When one mistakenly drink methanol, it may be helpful to drink
liquor (ethanol). The reason is that ethanol serves as the competitive
inhibitor for the dehydrogenation of methanol by ADH.
Problems
(1) Using the Michaelis–Menten equation, confirm that K m is equal
to the concentration of substrate to give the rate of V/2.
(2) For the Ping-Pong bi-bi mechanism, derive Eq. 3.5 and express
K 1 and K 2 by the rate constants.
(3) For the competitive inhibition, derive Eq. 3.8.
(4) For the uncompetitive inhibition, derive Eq. 3.12.
References 51
References
1. Cornish-Bowden, A. (1976). Estimation of rate constants in Principles

of Enzyme Kinetics. Betterworths. London, Boston. pp. 168–197.
2. Lineweaver, H., and Burk, D. (1934). The determination of enzyme
dissociation constants. J. Am. Chem. Soc., 56, 658–666.
3. Hanes, C. S. (1932). Studies on plant amylases. I. The effect of starch
concentration upon the velocity of hydrolysis by the amylase of
germinated barley. Biochem. J., 26, 1406–1421.
4. Eadie, G. S. (1942). The inhibition of cholinesterase by physostigmine
and prostigmine. J. Biol. Chem., 146, 85–93.
5. Haldane, J. B. S. (1957). Graphical methods in enzyme chemistry. Nature,
179, 832.
6. Cleland, W. W. (1963). The kinetics of enzyme-catalyzed reactions
with two or more substrates or products. I. Nomenclature and rate
equations. Biochim. Biophys. Acta, 67, 104–137.
7. Dworschack, R. T., and Plapp, B. V. (1977). Kinetics of native and
activated isoenzymes of horse liver alcohol dehydrogenase.
Biochemistry, 16, 111–116.
8. Pilloff, D., Dabovic, K., Romanowski, M. J., Bonanno, J. B., Doherty,
M., Burley, S. K., and Leyh, T. S. (2003). The kinetic mechanism of
phosphomevalonate kinase. J. Biol. Chem., 278, 4510–4515.
9. Nakamurs, S., and Ogura, Y. (1968). Action mechanism of glucose
oxidase from Aspergillus niger. J. Biochem., 63, 308–316.
10. Fromm, H. J. (1979). Use of inhibitors to study substrate binding
order. Methods Enzymol., 63, 467–486.
11. Cornish-Bowden, A. (1974). A simple graphical method for
determining the inhibition constants of mixed, uncompetitive and
non-competitive inhibitors. Biochem. J., 137, 143–144.
12. Nakamura, T. (1993). Enzyme Kinetics. Japan Scientific Soc. Press.
Tokyo, Japan (in Japanese).
13. Dixon, M., and Webb, E. D. (1958). Enzymes. Longmans, Green and
Co, London, New York, Toronto.
Chapter 4
Effect of pH, Temperature, and High

Pressure on Enzymatic Activity
Chapter 3 described the effect of enzyme, substrate, and inhibitor

concentrations on the enzyme activity. We experience that the
reaction rate changes with pH and increases with the increase of
temperature. This chapter deals with the effect of pH, temperature,
and pressure on the enzyme activity [1, 2].
4.1 Effect of pH
4.1.1 A Basic Model
Chapter 2 described the importance of pH on the enzyme activity.
The activity changes with changes in pH, and most enzymes show
the bell-shaped pattern of the activity-pH profile. The profile can be
simply explained by assuming that the enzyme has two ionizable
groups responsible for its activity. Increasing pH, the enzyme (EH2)
having two protons loses one proton to form the active enzyme
(EH), then finally loses another proton to form the inactive enzyme
(E): EH2  EH  E. Each enzyme species binds with substrate (S),
but only EHS is able to form its product (P). In addition to the
Michaelis–Menten mechanism, we consider the effect of pH on
enzymes, and introduce the following scheme (Eq. 4.1). In Eq. 4.1,
K denotes dissociation constant of each complex. In Eq. 4.2, H

Haruo Suzuki
www.panstanford.com
54 Effect of pH, Temperature, and High Pressure on Enzymatic Activity
represents the concentration of proton. Equilibrium steps are rapid

as compared with the irreversible step: EHS to EH + P, then the rate
(v) of reaction can be obtained.
(4.1)
EHH      
K e1 = , K e2 = E H , K es1 = EHS H , K es2 = ES H
EH2  EH EH2S EHS
[EH2 ][S]
K ¢s = , K s = [EH][S] , K ¢¢s = [E][S] (4.2)
[EH2S] [EHS] [ES]

The total concentration of enzyme, e0, is
e0 = [E] + [EH] + [EH2] + [ES] + [EHS] + [EH2S] (4.3)

From these equations, [EHS] is deduced, and introduced into
v = k+2[EHS] (4.4)
k+2e0
v= . (4.5)
1+ H/K es1 + K es2/H +(K s /[S]) (1+ H/Ke1 + K e2/H )
This simplifies to Eq. 4.6,
V[S]
v = __________
(4.6)
[S] + Km
Comparing Eq. 4.6 with Eq. 4.5, these equations are isomorphic;
then
Effect of pH 55
V
V= (4.7)
1+ H/K es1 + K es2/H
K s (1+ H/Ke1 + K e2/H ) (4.8)

Km =
V V / Ks
= (4.9)
K m 1+ H/K e1 + K e2/H
V 1
 =
k
where V = 1++2H0/,Kthe e es1 +pH-independent
K es2/H . maximum rate, and V, the
v=
1+ H/K es1maximum
+ K es2/H +(rate at various
K s /[S]) (1+ H/KpH. + KFrom /H )the pH-dependent changes of
K (1+ H/Ke1 K+s (1+
K e2/HH/K)e1e1 + Ke2e2/H )
V, K m, =ands V/K m, =the dissociation constants can be determined. It is
1+ H/K +1+ K es2
H/contains
H
K es1 + K es2the
/H proton dissociation constants
interesting that es1 Eq. 4.7
of the enzyme-substrate complex and Eq. 4.9 those of the free
K s (1+ HK/K(1+
enzyme. e1 +H K/e2K/e1H+) K e2/H )
Km = Km = s
1+ H/K1+ and
es1 +HK/K /H
es2es1are the
+ K es2 /Hproton dissociation constants of the enzyme
and of the enzyme-substrate complex, respectively. When the
K (1+ H/Ke1of
deprotonation + Kthe /H K) (1+site
active H/Kresidue
e1 + K e2/is H )affected by binding with
Km = s Ke2m = s
substrate,
1+ H/K es1is+ not K /equalK (1+
H HH//KKes1
1+ to . ++
e1 KKK s (1+
However,//HH)H/when
Ke1 + Kite2/isH )not affected
K es2= s K = e2es2
with the substratem binding, 1+ H/K es1ism+equal K es21+/HtoH/K es1. +
InKaddition
es2/H to these,
functional groups in the active site of enzyme would be speculated
from the proton dissociation constant.
4.1.2 Graphical Methods to Determine pK Value

Dixon plot is usually used to determineK spK (1+
K (1+
H/KHe1/K
values +[1].
K+e2K
e1 /From
He2/
) H )Eqs.
K m K=m = s
4.7 and 4.9, the proton dissociation constants,
1+ 1+
H/KHes1
/K+ and (1+
K+es2K/es2
sH HH/Ke1 + K e2/H )
,/and
Kes1
m =
K s (1+ H/and
Ke1 + K e2,/Hrespectively,
) are determined. With rearrangement 1+ H/K es1of+ K es2/H
Km =
1+ H/KEqs. 4.7 and 4.9, we have
es1 + K es2/H
V 1
= (4.10)
V 1+ H/K es1 + K es2/H

(V/K m ) 1
 (4.11)

(V/K s ) 1 + H/K e1  K e2/H

Both Eqs. 4.10 and 4.11 are isomorphic.
V 1
Therefore, Eq. 4.10 is used
=
to explain the Dixon plot. Log (V/V ) is1+
plotted
H/K es1against
+ K es2/HpH (Fig. 4.1).
The following explanation can be applied for the corresponding plot
for Eq. 4.11. Here, the acidic and alkaline pK are represented as pK1
and pK2, respectively. Increasing pH, the crossing point between
horizontal and vertical guidelines with a slope of 1 gives an acidic
pK1 (Fig. 4.1; pKes1, pKe1 of Eqs. 4.10 and 4.11, respectively). Similarly,
Log (V/ V ) 1+ H/K es1 + K es2/H

we get an alkaline pK2.
1
V
=
V 1
=
Figure 4.1 Dixon plot. Log (V/ V ) values
1+ H/Kare plotted against pH. The
es1 + K es2/H
theoretical values obtained with the constant pK1 = 6 and
various pK2 = 7 (line), 8 (dashed line), and 9 (short dashed
line) are shown. Guidelines are shown by thin dashed line.
Crossing points are the pK values to be determined.
Figure 4.1 shows Dixon plots of a hypothetical enzymatic

reaction. Three cases of the enzymatic reaction having the pK1 of 6
and three different pK2 of 7, 8, and 9, are assumed. The plots give
pK1 values deviated from the assumed pK values, especially in the
combination of pK1 = 6 and pK2 = 7 (see Fig. 4.1). Similarly, an alkaline
pK2 is also deviated from an assumed pK2. Interestingly, VpK values 1
=
at the crossing point with the horizontal line of log (V/V ) =0 1+are
H/K es1 + K es2/H
agreed well with assumed pK values, respectively. Here, it is
recommended to use the Dixon plot in keeping in mind with
these facts. Simply, apply the Dixon plot to the case that the
difference between pK1 and pK2 is assumed to be greater than 3.
The next method Vis to determine 1 pH values that give the half
maximum value of V/VV  . =pH and
1+1 H 1+2Kare
pH
/K es1 the pH values at the half
= es2/H
maximum value of V/V . As 1+Fig.
H/K4.2es1 +shows,
K es2 H
/ pH1 of the acidic side
is deviated from the assumed pK1 = 6, especially for the combination
of pK1 = 6 and, pK2 = 7 and 8. Similarly, pH2 values at the alkaline
Effect of pH 57
side are deviated from the assumed pK2 values, especially for
the combination of pK1 = 6 and pK2 = 7 and 8. These analyses
recommend to use the method when the difference of pK
(=pK2 – pK1) is assumed to be greater than 3.
The third method is to determine pK values applicable for
the cases that the difference in the pK values is smaller than 3.
Based on the plot of Fig. 4.2, determine pHop, pH1, and pH2. Then,
the proton concentration at these pHs are calculated to be [H+o p ] ,
[H 1+ ], and [H +2] , respectively. These concentrations are represented
as Hop, H1, and H2, respectively. Then from Eq. 4.7, we have
K1K2 = (Hop)2 (4.12)
H1 + H2 = 4Hop + K1 (4.13)
Hop, H1, and H2 can be determined from the plot (Fig. 4.2); then it
is easy to calculate pK1 and pK2. For example, let us determine pK
values from the plot in Fig. 4.2. Try with the smallest bell-shaped
line that was obtained by assuming pK1 = 6 and pK2 = 7. By enlarging
Fig. 4.2, the pHop, pH1, and pH2 were estimated to be 6.50, 5.64,
and 7.32. Then, we get the values: Hop = 3.16 × 10–7 M, H1 = 2.9 ×
10–6 M, H2 = 4.79 × 10–8 M.

1
=
V/ V
V
V 1
 =
Figure 4.2 V/ V vs.1+pH
H/Kplot for the hypothetical enzymatic reaction
es1 + K es2/H
V in Fig. 4.1.
shown 1 Horizontal dashed lines show half of the
=
V/ V values
1+ H/atK es1
each+ Koptimum
es2/H
V pH. This method
=
1 indicates that
the crossing point with V/ V value
1+ Hgives
/K es1 the
+ K es2 /H (pK1) and
acidic
alkaline pK (pK2).
Introducing these values to Eq. 4.13, we can getpK 1 = 1.08 × 10–6 M,

and pK 1 = 5.97, which agrees well with the assumed pK 1 = 6. Then
pK 2 is calculated from Eq. 4.12 to be 9.25 × 10–8 M, and pK 2 is
calculated to be 7.03. The same treatments of the kinetic data are
applicable for Eq. 4.11. Thus, we can get pK s values of ionizable
groups in the enzyme itself and the enzyme-substrate complex.
4.1.3 Meaning of pK Values

The pK values of the kinetically influential ionization can be
determined as described above. As Table 4.1 shows, ionizable
groups in the enzyme have different pK a values. Therefore, by
comparing the experimentally determined pK a values with those in
Table 4.1, the ionizable group in the enzyme could be identified.
However, it is not so simple because ionization of groups in the
enzyme is affected by the microenvironment of the groups. For
example, carboxy and amine groups have higher and lower pK a,
respectively, in hydrophobic environments than those in hydrophilic
environments [3, 4].
Table 4.1 pKa of ionizable group in amino acid residues
Ionizable groups pKa

Carboxy 2~6
Imidazole 5.6 ~ 7.0
Sulfhydryl 7.5 ~ 10.3
Phenolic hydroxy 9.1 ~ 10.8
Amino 10 ~13
Guanidino 12~ 13
Source: From [2] with permission.
In the case of pK e1 = pK es1, and pK e2 = pK es2, it means that

deprotonation of ionizable groups in the enzyme is not affected
by binding with substrate. This suggests that deprotonation of the
ionizable group is not involved in the binding of substrate, thus
the group is not related to the substrate binding. In the other case:
pK e1 ≠ pK es1, and pK e2 ≠ pK es2, which means that deprotonation
of the ionizable group is involved in the binding of substrate.
Thermodynamics in the Enzymatic Reaction 59
Figure 4.3 shows the example of this type of ionization. The

baker’s yeast l-lactate dehydrogenase (cytochrome b2) catalyzes
the dehydrogenation of l-lactate to form pyruvate. The pK a values
obtained suggest that the ionizable group with pKe1 = 6.0 becomes
easily deprotonated by binding with substrate, since pK es1 is 5.3.
Similarly, at alkaline side, the deprotonation of ionizable group
with pK e2 = 8.8 is perturbed by binding with substrate, since pK es2
is 9.6. That is, the protonated form is stabilized by binding with
substrate. From the pK a values, it was suggested that His residue and
amino group were proposed to be the ionizing groups in acidic and
alkaline sides, respectively [5]. Later, structural studies revealed
that His373 and Tyr254 are in the active site of the enzyme [6].
Figure 4.3 Dixon plot for the yeast l-lactate dehydrogenase (cytochrome
b2) reaction. Reproduced from Suzuki and Ogura [5].
4.2 Thermodynamics in the Enzymatic Reaction

We know with our experience that physical and chemical
properties of substance change with the change in temperature.
Before entering the effect of temperature on the enzymatic reaction,

let us start by summarizing the basis of thermodynamics, since
thermodynamics is useful to understand the effect of temperature
on the enzyme-catalyzed reaction.
4.2.1 Basics of Thermodynamics

The first equilibrium step in the Michaelis–Menten mechanism
is assumed to be a rapid process as compared with the second
irreversible one,

E+S  ES 
  E+ P (4.14)
The equilibrium constant, K, is expressed as follows:
[ES]e
K = _________
(4.15)
[E]e[S]e
where [E]e, [S]e, and [ES]e represent the equilibrium concentration

of E, S, and ES, respectively. The change of the standard free
energy (DG°) is a function of the equilibrium constant.
DG  = –RT ln K (4.16)
where T is an absolute temperature and R the gas constant.
The equilibrium constant K is related to the standard enthalpy
change (DH°) for a given reaction, known as van’t Hoff equation:
d ln K DH  (4.17)
=
dT RT 2
By the determination of K values at various temperatures, the
slope of a linear plot of ln K vs. 1/T will give the DH° value.
The second law of thermodynamics gives Eq. 4.18:
DG = DH – TDS (4.18)
where DS represents the entropy change. These give us three

thermodynamic parameters for the equilibrium: E+S 
 ES , 
  E+ P
DG°,
DH°, and DS°.
The entropy (S) is expressed according to the Boltzmann
equation:
S = kB ln W (4.19)
–1
where kB is the Boltzmann constant 1.3807 × 10–23 JK ; obtained
by dividing gas constant by Avogadro constant), and W is the
number of microstate of a system. The concept of W is difficult to
explain. It may not be strict, but a generally accepted meaning of
entropy is a measure of randomness or uncertainty of a system.
From Eq. 4.20, DS° can be determined. Thus, we have three
thermodynamic parameters, DG°, DH°, and DS°.
D H  – DG 
DS   (4.20)
T
Box 4.1
Standard State, Unit of Pressure, Gas Constant
It is also called the thermodynamic standard state. To write the

thermodynamic parameters, we need the standard conditions.
Pure gas, pure solid under the pressure of 1 bar are defined to be
in the standard state. As for a solution, 1 molar (M) of pure solute
is defined to be standard state of solution. The standard state is
expressed by superscript, “°”. You may find the example in the text.
Temperature is not included in the definition, but it is usual to
use 25°C (298.15 K) [or 0°C (273.15 K)]. Here, the standard state
of 1 M H+ is too acidic in the biological field, so the neutral pH 7.0
(10–7 M H+) is used for the standard state and is called “biochemical
standard state.”
As for the unit of pressure, Pa (pascal) is now internationally used.
This unit is named from the “the Principle of Pascal.” The relationship
of pressure with its unit,
1 bar = 105 Pa = 102 kPa = 0.987 atm
The relationship of gas constant with its unit,
R = 8.314 J K–1 mol–1 = 1.987 cal deg–1 mol–1 = 0.0831 L bar K–1 mol–1.
4.2.2 Transition State Theory

A most popular theory to explain the kinetics of reaction is the
transition state theory (1935 by Eyring, and by Evans and Polani)
[7, 8]. The theory was applied well for enzyme-catalyzed reaction
by Pauling (1948) [9]. The active site of an enzyme is precisely
complementary to the reactants in their activated transition state

geometry. Thus, an enzyme strongly binds to the transition state
molecule and greatly increases its concentration, and thereby
accelerates the reaction proportionately [9]. This idea is extended
to produce abzyme or in other word, catalytic antibody, which has
produced against the transition state analogue closely resembled
the transition state molecule (Jencks, 1969) [7]. Most of the
abzymes produced, however, have the low catalytic activity. This
fact implies that the active site complementary to the transition
state is not sufficient for the rate enhancement of enzyme.
Now the structural studies of various enzymes reveal that each
enzyme has its own structure of enzyme, that is, there are acidic,
alkaline residues, metals, or prosthetic group in the active site.
Moreover, the hydrogen (proton, hydride, or hydrogen atom)
transfer reaction may occur by the quantum tunnel (through
barrier) as shown by the dashed arrow in Fig. 4.4. The example
of the hydrogen transfer will be described in Chapter 10.
Figure 4.4 Energy profiles of uncatalyzed and enzyme-catalyzed reactions.

DG≠S > DG≠ES. The dashed arrow indicates a quantum tunnel
through barrier.
For the analysis of the enzymatic reaction, useful relationship will

be introduced. The substrate S must pass over an energy barrier
between an initial and a final state. The substrate changes to an
unstable state (transition state), S≠ to form the product P:

S S P
 (4.21)
The transition state

S  (S)
 isinP equilibrium with the initial state.
Then,
[S ] (4.22)
K =
[S]
DG = –RT ln K  (4.23)
DG =where
–RT ln K  and DG =are–RTthe equilibrium
ln K 
constants in Eq. 4.21 and
the free energy of activation to reach the transition state, respectively.
The concentration of substrate in a transition state is
 –DG 
[S ]=[S] exp  (4.24)
 RT 
The rate of the product formation (v) is dependent on the frequency

of the decay of the transition state:
kBT  (4.25)
v= [S ]
h
where h is the Planck constant (6.626 × 10–34 J s) and kBTthe

–1 v= [S ]
Boltzmann constant 1.3807 × 10–23 JK . From Eqs. 4.23, and 4.24,
h
kBT  –DG 
v= [S] exp  (4.26)
h  RT 
As the free energy of activation is from Eq. 4.18,
DG = DH  – T DS 
Then, the first order rate constant of the disintegration

DG = DH  – TofDS , k f , is
kBT  –DG  kBT  – DH    DS  

kf = exp = exp exp  (4.27)
h  RT  h  RT   R 
This equation has been known as the Eyring equation. Differentiation

of the natural logarithm of Eq. 4.27 gives
d ln kf DH  + RT
= (4.28)
dT RT 2
From the Arrhenius Eq. 1.1 and Eq. 4.28,
DH≠ = Ea – RT (4.29)
  kBT 
The activation free energy, DG is
= –from
RT ln kf 4.27,
Eq. – ln 
  h 
  kBT  (4.30)
DG = –RT ln kf – ln 
  h 
4.2.3 Determination of Thermodynamic Parameters of

the Enzymatic Reaction
When the reactions follow the Michaelis–Menten mechanism, the
energy change with the reaction co-ordinate will be written in
Fig. 4.4.
The step: E+S    ES   E+ P
From Eq. 4.16, we have the standard free energy using the
[EH2 ][S]
dissociation constant of theKES ¢s = complex, , K ,= [EH][S] , K ¢¢ = [E][S]
[EH2S] s [EHS] s [ES]
[EH2 ][S] [EH][S] , [E][S]
¢ ,
= = RT ln K s =
K s DG° K =
¢¢ (4.31)
[EH2S] [EHS] s [ES]
Here, “–” is not written before R in Eq. 4.31 as compared with
Eq. 4.16. This is because the equilibrium constant K is the reciprocal
[EH2 ][S] [EH][S] , [E][S]
of the dissociation =constant
K ¢s H , K s.=Assuming =
K ¢¢s that the Michaelis
K s (1+ /K
[EH + K [EH
e1 S]e2 2 / H )
][S] [EH][S] [E][S]
constant, K m is
= approximately 2 equal to[EHS]
K ¢s = , K s,= ,[ES]
K ¢¢s =
1+ H/K es1 + K es2[EH /H 2S] [EHS] [ES]
DG° = RT lnKm (4.32)
Following Eq. 4.17, we get
d DH 
(ln K m ) = – (4.33)
dT RT 2
K s (1+ H/Ke1 + K e2/H )
The enthalpy change can be determined by measuring K m at
= various
1+ H/K + K es2/H
temperatures. That is, the thermodynamic parameters of the es1
equilibrium: E+S 
 ES can
beE+
determined.
P
Temperature Dependence of the Enzymatic Reaction 65
The step: ES  ES ‡

From the temperature change of kcat of the enzymatic reaction,
we get the activation energy, E a, using Arrhenius equation, Eq. 1.2.
Then the activation enthalpy can be calculated (Eq. 4.29).
Moreover, we can determine the activation energy (Eq. 4.30), thus
we can determine the thermodynamic marameters of activation:
GDG
DD G=, =D
 
=DH
DHH,–and
 
–T–TD
TDSDSS.
4.3 Temperature Dependence of the Enzymatic

Reaction
The rate of the enzyme-catalyzed reaction increases with
increasing temperature. However, at higher temperature, the rate
decreases gradually. Figure 4.5a shows a hypothetical enzyme-
catalyzed reaction at various temperatures. At 20 and 30°C, the
product is formed linearly with time, but at higher temperature
the product formation decreases with time. As Fig. 4.5b shows, the
initial rate of the product formation has an optimum temperature
like a pH-activity profile. The decrease of the rate at higher
temperature must be due to the heat denaturation of enzyme.
Therefore, an optimum temperature is derived from the balance of
the increase and decrease of the rate. This means that the maximum
rate observed at the optimum temperature (40°C in Fig. 4.5) is the
rate observed with the mixture of the active and inactive forms of
enzyme. Thus, for the kinetic analysis of enzyme, we should measure
the enzyme activity sufficiently below an optimum temperature
not to estimate the rate with the mixture of active and inactive
enzymes. To perform the experiments without inactive enzyme,
the experiments similar to the test of the reversibility of
inhibition are recommended (see Chapter 3). After the enzyme is
pre-incubated at given temperature for given times (for example,
10 min), the whole is cooled in the ice-cold water. Then the enzyme
activity is measured at lower temperature, for example, at 25°C,
using a part of the pre-incubated enzyme. Then the residual
activity is plotted against temperature. In the case of the enzyme
shown in Fig. 4.6, the kinetic analysis is better to be done at or
below 30°C.
(a) (b)
Figure 4.5 Effect of temperature on the hypothetical enzyme reaction.

(a) Time-dependent product formation. (b) Temperature
dependency of initial rate. Product formed and initial rate are
arbitrary unit.
Figure 4.6 Heat stability of enzyme. An enzyme was preincubated at

given temperatures for 10 min. Then the whole was cooled,
and the enzyme activity was assayed at 25°C. The enzyme
activity at various temperature was divided by the activity
measured at 25°C. The value (%) was plotted against
temperature.
4.4 Effect of Pressure

The high pressure affects on the enzyme activity, and most enzymes
are reversibly inactivated over 3 kbar, and irreversibly over
7 kbar [10, 11]. Therefore, kinetic analysis is recommended to be
performed under the conditions without inactivated enzymes.
Effect of Pressure 67
4.4.1 Effect of Pressure on the Rate of Reaction

The reactant state is in equilibrium with the transition state
as described in the previous section. Applying the equilibrium
thermodynamics to the quasi-equilibrium between the reactant
state and the transition state (Eq. 4.21), we have the following
relation at a constant temperature:
d ln K  DV  (4.35)
=–
dp RT
d lnwhere
K DV  is the activation volume, and defined to be the difference
=–
dpbetweenRTthe volume of the transition state and that of the reactant
state. Substitution of Eq. 4.23 gives
d( DG )
= DV  (4.36)
dp
Applying Eq. 4.36 to 4.27, and rearrangement, we have
d ln k d kBT DV  DV 
= ln – =– (4.37)
dp dp h RT RT
Thus, we can determine the activation volume by measuring the

rate of enzymatic reaction at various pressures.
4.4.2 Meaning of the Activation Volume

There are many examples of the determination of the activation
volume. The 104 values of the activation volume ranging from
–69 to 175 cm3/mol were reported at the time of 1986 [10]. The
pressure affects various interactions in the enzyme protein, and
the interactions of enzyme with substrate. The interactions are
ionic, hydrophobic, electrostatic (hydrogen bonding), and van der
Waals interactions. Moreover, the behavior of water molecule is
also affected by pressure. Therefore, it is still vague to conclude
what interactions determine the activation volume. Those who are
interested in the field may find the references in this chapter. The
next section describes how temperature and pressure affect on
the enzymatic reaction [10, 11].
4.5 The Effect of Temperature and Pressure on

α-Chymotrypsin-Catalyzed Reaction
a-Chymotrypsin (a-CHT) is the protease that catalyzes the hydrolysis
of the peptide bond at the C-terminal side of hydrophobic amino
acid residues. a-CHT is expressed as a proform, chymotrypsinogen,
which is proteolytically activated to produce its final form. a-CHT
is composed of three subunits, a, b, and c, and these subunits are
linked by the disulfide bonds. a-CHT also catalyzes the hydrolysis
E + bond. The catalytic mechanism of the enzyme is,
of the esterS 

 k+3
ES k+2
E+S 
 ES    ES¢   E+ P2 (4.38)

P
ES1¢


E
(a) (b)+ P2
Figure 4.7 3D structure of α-CHT-transition state analog complex.

(a) The surface view of overall structure of monomer. TS
analog (phenylethane borate, PBA: yellow cubic model) is
covalently bound to the side chain O atom of Ser 195. b subunit,
green; c subunit, magenta. a subunit is behind these subunits
in this Figure. The phenyl group of PBA is in the pocket of
α-CHT. (b) Close-up view of the binding site of PBA. The
numbers near the dashed line is the length (Ǻ) of the hydrogen
bond. The figure is drawn from the pdb 6cha using PyMOL.
where ES¢ represents the acylated intermediate of enzyme [12].

The catalytic site of enzyme contains Ser-195, His-57, and Asp-102,
and these residues form the charge relay system, which induces
the negative charge on the side chain oxygen of Ser-195. Thus,
the oxygen atom of Ser-195 nucleophylically attacks the carbonyl
The Effect of Temperature and Pressure on α-Chymotrypsin-Catalyzed Reaction 69
carbon of the peptide bond of substrate protein to produce the

acylated enzyme. The amide nitrogen in the protein accepts pro-
ton from His-57 and dissociates from the enzyme. His-57 receives a
proton from nearby H2O, and the resulting OH– attacks the carbo-
nyl carbon of the acylated enzyme to release the acyl group from
the enzyme to reform the original enzyme, α-CHT. The tetrahedral
transition state intermediate is assumed to be formed at the step
formation of the acylated enzyme: ES to ES. The X-ray crystal-
lographic analysis of the enzyme in complex with the transition
state analog, phenylethane boric acid revealed that the amino acid
residues are arranged well to explain the above mechanism, and
the analog binds covalently with the side chain oxygen atom of the
Ser-195 (Fig. 4.7).
4.5.1 Ef fect of Temperature

Bender et al. (1964) prepared four different acyl-enzymes, and
measured the rate of deacylation (k+3) at various temperatures [13].
The k+3 value of the N-acetyl-l-tyrosyl-enzyme is 3540 times greater
than that of the acetyl-enzyme. They determined the activation
parameters (Table 4.2). The activation enthalpy (DH≠) of the
deacylation is around 45 kJ/mol, but the activation entropy (DS≠)
decreased greatly with the change from N-acetyl-l-tyrosyl- to acetyl-
enzyme. That is, DS≠ is –56.1 e.u. (J K–1 mol–1) for N-acetyl-l-tyrosyl-,
and –150 e.u. for the acetyl-enzyme. The results suggest that the
difference in the large k+3 value is derived not from the change in
the enthalpy of activation, but that in the entropy of activation. It is
reasonable to assume that the entropy of TS is the same value for
the acyl-enzymes used. Then, the change of the entropy of activation
for the above acyl-enzymes, N-acetyl-l-tyrosyl- and acetyl-enzymes,
can be visualized (Fig. 4.8). The entropy is, as described in Eq. 4.19,
the measure of randomness of the system. Figure 4.8 clearly shows
that the acetyl-enzyme has higher value of entropy of activation
than the N-acetyl-l-Tyr-enzyme, suggesting that the acetyl group in
the acetyl-enzyme is movable than the acyl group in the N-acetyl-
l-Tyr-enzyme. The acyl group (RCO-) is bound to the hydroxyl
group of Ser195. The bond between the carbonyl C of RCO and the
hydroxyl O atom of Ser195 in TS must have a conformation to be
attacked by His57 (Fig. 4.7). In the case of N-acetyl-l-Tyr-enzyme,
the change in the entropy of activation is small, suggesting that the

acyl group in the initial state is well trapped in the active site of
the enzyme. On the other hand, the acetyl group is easy to move;
so the group is not trapped in the initial state; thus, the group is
movable. The difference in the activation entropy is approximately
100 J mol–1 K–1. Assuming that the rotational entropy per bond is
25 J mol–1 K–1, then 100 J mol–1 K–1 corresponds to the summation of
the rotational entropy of four bond, a-C (Ser195)-C-O-C(O)-R.
Table 4.2 Activation parameters for the deacylation of the acylated

α-CHTs
Acylated α-CHT DH≠ (kJmol-1) DS≠ (J mol-1/K–1)

N-Aceyl–l-tyrosyl- 43.1 –56.1
N-Aceyl–l-tryptophanyl- 50.2 –82.8
trans-Cinnamoyl- 46.9 –124
Acetyl- 40.6 –150
Source: From Bender et al. J. Am. Chem. Soc., 86, 1964, 3714–3721.
Figure 4.8 The entropy change from the initial state to the transition state
(TS) in the deacylation reaction of the acylated α-CHT. The
data in Table 4.2 are used. It is assumed in the figure that the
entropy of TS is the same for these acyl-enzymes.
The Effect of Temperature and Pressure on α-Chymotrypsin-Catalyzed Reaction 71
4.5.2 Effect of Pressure

The α-CHT-catalyzed reaction shown in Eq. 4.38 is modified to
Eq. 4.39 by adding tetrahedral intermediates in the acylation and
deacylation reactions.
E+
S 


Ks k +1 ES k +2 k +3
 ES 
E+S  
k–1  ET    ES¢  ET¢ 
  E+ P2 (4.39)

P
ES1¢


where ET and ET represent the tetrahedral E+ intermediates.
P
Makimoto et al. (1986) determined the 2activation volumes of
the acylation and deacylation reactions [14]. The following is
the summary of the studies on the acylation reaction. Using
p-nitrophenyl pivalate (pNPT) as substrate (Fig. 4.9), the rate of the
acylation reaction was determined under various pressure, ranging
from 1 bar to 1.8 kbar at pH 7.8 and 25°C. Figure 4.10 shows the
rate of acylation vs. pressure.
Figure 4.9 Structure of p-nitrophenyl pivalate.
Figure 4.10 Determination of the activation volume of the acylation

reaction. pH 7.8 and 25°C. The data in ref. 14 were used. The
slope of the line is 0.415 kbar–1.
Using Eq. 4.38, the slope of the linear plot gives the activation
volume of the acylation reaction. The volume is calculated to be
d ln k d kBT DV  = –23.7
DV  cm3/mol using the gas constant of 0.0831 L bar
= ln – = –
dp dp h K–1mol–1
RT . The rate-determining step of acylation reaction for the
RT
k1
p-nitrophenyl ester substrate is known to be the step: ES   ET.
kBT DV  = –23.7
ln k d volume
Therefore, the dactivation DV  cm3/mol must be
= ln – =–
explained by the dp
interactions
dp hin theRT
transition
RT state (Fig. 4.11). The
interactions are,  the formation of the covalent bond between the
O atom of Ser195 and the carbonyl C of substrate,  the charge
concentration at the carboxy O atom of substrate and at the N
atom of the imidazole ring of His-57, and the formation of two
hydrogen bonds  between the O atomof Ser195 and the N atom
of the imidazole ring, and  between the N atom of the imidazole
and the carboxy O atom of Asp102. These interactions are
considered to be formed simultaneously [14]. From the model
system [see 14 for references], the volume changes are known.
That is, –10 cm3/mol for the formation of covalent bond, –5 cm3/
mol for the hydrogen bonding, and –5.5 cm3/mol for the charge
concentration. The sum of these values (–25.5 cm3/mol) agrees
with the activation volume (–23.7 cm3/mol). Therefore, these
interactions are in the transition state from ES to ET.
Figure 4.11 Change of ES to ET in the acylation reaction of αCHT. TS

represents the transition state. Reproduced with permission
from Makimoto et al. Bull. Chem. Soc. Jpn, 1986, 59, 243–247
with permission.
Problems
(1) Derive Eqs. 4.12 and 4.13.
(2) Bender et al. studied the deacylation reaction of N-acetyl-l-
tyrosyl-α-chymotrypsin. They obtained k+3 = 193 s–1, and the
References 73
activation enthalpy, 10.3 kcal/mol (45 kJ/mol). Confirm the

activation entropy (–56.1 e.u.) described in Table 4.2.
(3) Confirm the activation volume calculated in Section 4.5.2.
References
1. Dixon, M., and Webb, E. C. (1958). Enzymes. Longmans, Green and Co.
London, New York, Toronto.
2. Nakamura, T. (1993). Enzyme Kinetics. Japan Scientific Societies Press
(in Japanese).
3. Harris, T. K., and Turner, G. J. (2002). Structural basis of perturbed
pKa values of catalytic groups in enzyme active sites. IUBMB Life, 53,
85–98 (review).
4. Isom, D. G., Castañeda, C. A., Cannon, B. R., Velu, P. D., and Garcis-
Moreno, E. (2010). Charges in the hydrophobic interior of proteins.
Proc. Natl. Acad. Sci. USA, 107, 16096–16100.
5. Suzuki H., and Ogura, Y. (1970). Effect of pH on the kinetic parameters
of yeast l(+)-lactate dehydrogenase (cytochrome b2) J. Biochem.,
67, 291–295.
6. Xia, Z.-X., and Mathews, F. S. (1990). Molecular structure of
flavocytoshrome b2 at 2.4 Å resolution, J. Mol. Biol., 212, 837–863.
7. Jencks, W. P. (1969). Catalysis in Chemistry and Enzymology. McGraw-
Hill, New York.
8. Laidler, K. J., and King M. C. (1983). The development of transition-
state theory. J. Phys. Chem., 87, 2657–2664 (review).
9. Pauling, L. (1948). Chemical achievement and hope for the future.
Am. Scientist, 36, 51–58.
10. Morild, E. (1981). The theory of pressure effects on enzymes.
Adv. Protein Chem. 34, 93–166 (review).
11. Boonyaratanakornkit, B. B., Park, C, B., and Clark, (2002). Pressure
effects on intra-and intermolecular interactions within proteins.
Biochim. Biophys. Acta, 1595, 235––249 (review).
12. Blow, B. M., Birktof, J. J., and Hartley, B. S. (1969). Role of a buried
acid group in the mechanism of action of chymotrypsin. Nature, 221,
337–340.
13. Bender, M. L., Kezdy, F. J., and Gunter, C. R. (1964). The anatomy
of an enzymatic catalysis a-chymotrypsin. J. Am. Chem. Soc., 86,
3714–3721.
14. Makimoto, S., Suzuki, K., and Taniguchi, Y. (1986). Effect of pressure
on the pre-steady state kinetics of the hydrolysis of p-nitrophenyl
pivalate catalyzed by a-chymotrypsin. Bull. Chem. Soc. Jpn., 59,
243–247.
Chapter 5
Measurement of Individual Rate

Constants
Chapter 2 described the Michaelis–Menten mechanism of the

enzymatic reaction:
E+S 
 ES  E + P
The overall reaction kinetics tells that the maximum rate is
equal to k+2e0. Thus, we can easily determine the rate constant k+2
dividing the maximum rate by the total enzyme concentration.
However, it is not certain whether the rate constant is correct
without observing directly the step: ES to E + P. As described in
Chapter 2 (Section 2.3.2), the time scale of the step is usually below
1 sec. Therefore, the special apparatus is required to observe the
step. The readers may recall one of the stopped-flow methods in
Chapter 2. This chapter describes the method in detail and focuses
on the analysis of the first-order reaction.
5.1 Rapid-Mixing Techniques

Hartridge and Roughton (1923) used the flow method for the first
time to study on the liquid reaction [1]. They invented the method

Haruo Suzuki
www.panstanford.com
76 Measurement of Individual Rate Constants
to detect a reaction with a half-life of 3 ms and used it to measure

the rate of binding of ligands with hemoglobin. The principle of
the method is shown in Fig. 5.1. One syringe contains enzyme and
the other substrate. Then both solutions are mixed by pushing
the top of the syringes. The enzyme and substrate are mixed well
by the mixing chamber, and travel the flow tube. At various points
of the tube, the reaction can be monitored. At the constant flow
of the mixed reactants, the time after mixing can be changed
by observing the reaction at various distances from the mixer.
The problem of the method is that a lot of enzyme is required to
reach the constant signal of the detector. For the study on the
enzyme, it is not easy to prepare sufficient amounts of highly
purified enzyme. Chance (1940) invented the apparatus available
for the small amounts of reactants and used it to analyze the
peroxidase reaction [2]. Currently, 0.05 to 0.2 mL for each may be
required for one mixing.
Figure 5.1 The schematic view of the continuous-flow method. M (circle)

represents a mixing chamber. Blocked arrow shows that the
position of the monitor (D) is movable upward or downward
to change the time from M.
The methods mentioned above are included in the rapid-mixing

methods (techniques). The various detectors have been invented
to observe the reaction after mixing (Table 5.1). The method
is classified into the continuous-flow method and stopped-flow
Rapid-Mixing Techniques 77
method. The continuous flow method observes the reaction during

the flow of reactant liquid as shown in Fig. 5.1. The stopped-
flow method observes the reaction after the flow stops, and the
apparatus for this method is supplied by scientific instrument
companies.
Table 5.1 Monitors in the rapid-mixing methods.
Groups Type of monitor

Spectroscopy Absorbance; fluorescence; circular dichroism (CD)
Heat production Thermocouple
Magnetic Resonance Electron paramagnetic resonance (EPR)
Radioactivity Liquid scintillation counting
The first point to notice in the rapid-mixing method is the

dead time that is the time required for the reactants to reach the
detector after mixing. The time of the usual apparatus is 0.5 to 2 ms.
This means that the reactions finished in the dead time are not
observable. For example, for the reaction of the first-order rate
constant of 350 s–1, we can only monitor the remaining half of the
reaction by using the apparatus with the dead time of 2 ms, since
the half-life is calculated to be 2 ms using Eq. 5.8.
The second point is the response of the detector. In the case
of the slow response of monitor, reactions are usually stopped at
given times after mixing, and the progress of reaction is measured
by using an appropriate detector. As the response of detectors of
EPR and radioactivity is slow, relatively longer times are required
for the measurement. Bray invented the rapid-freezing method for
the EPR measurements (schematically shown in Fig. 5.2a) [3]. At
given times after mixing the reactants, the reaction mixtures were
quickly frozen by flushing the reaction mixture into the liquid
isopentane (mp: –160°C) cooled with liquid nitrogen, and the EPR
was measured. To change the time after mixing, the length of the
tube from the mixer to isopentane was changed. Cytochrome
b2 (l-lactate dehydrogenase of Baker’s yeast) catalyzes the
dehydrogenation of l-lactate to pyruvate using an electron acceptor
such as cytochrome c or authentic compounds (ferricyanide or
methylene blue). The enzyme contains FMN and cytochrome b2 as
its prosthetic groups. The FMN cofactor dehydrogenates l-lactate,

and one electron transfers from the reduced FMN to cytochrome
b2. The oxidized cytochrome b2 is one electron acceptor, therefore
the reduced FMN loses one electron to form half-reduced FMN
(FMN semiquinone). This mechanism of the cofactor reduction was
supported by the rapid-freezing technique [4]. Figure 5.2 shows
the semiquinone level of cytochrome b2 detected. The rapid
formation of FMN semiquinone agrees with the rate of reduction
of these prosthetic groups with l-lactate.
(a) (b)
Figure 5.2 (a) The schematic view of the rapid-freezing apparatus. The
enzyme (E) and substrate (S) are mixed by the chamber (M)
from where the reaction mixture is sent through the length-
changeable tube to the isopentane cooled with liquid nitrogen.
The fine particles of the mixture were pushed down to the
bottom of the EPR quartz tube. (b) The FMN semiquinone level
during the reduction of the FMN cofactor of yeast cytochrome
b2. Reproduced from Suzuki and Ogura [4].
For the detection of the radioactivity, the reaction mixture

obtained by the rapid mixing was further mixed with the denaturant
to stop the reactions. Then the radioactivity of the reactants was
measured (Fig. 5.3). Fersht and Jakes (1975) invented the apparatus
and used it to study on the Tyr-tRNA synthetase reaction:
+1k
E + Tyr + ATP   ETyr AMP + Pyrophosphate
+2k
ETyr  AMP + tRNA  E + Tyr-tRNA + AMP
They observed the amount of 32P-labeled ATP consumed as the

amount of E-Tyr~AMP formed as shown in Fig. 5.3 [5].
Analysis of the First-Order Reaction 79
(a) (b)
Figure 5.3 (a) The schematic view of the quenched-stopped flow

apparatus. Enzyme and substrate are mixed by the first mixer,
then the reactants are mixed with quencher by the second
mixer. The quenched reactants are collected to measure the
extent of reaction. The time after mixing at the first mixer is
controlled by changing the length of tube between the first
and second mixers. (b) The amount of Tyr-adenylate was
measured as that of ATP hydrolyzed in the partial reaction of
Tyr-tRNA synthetase (adapted with permission from Fersht,
A. R., and Jakes, R. Demonstration of two reaction pathways
for the aminoacylation of tRNA. Application of the pulsed,
quenched flow technique. Biochemistry, 14, 3350–3356.
Copyright (1975) American Chemical Society).
5.2 Analysis of the First-Order Reaction

This section describes how to determine the rate constant of the
first-order reaction. In various cases in enzymology, reactions can
be analyzed by the first-order reaction, like the pseudo-first-order
treatment of a second-order reaction. The more complex and
higher-order reactions are described in ref. [6] and in refs. [2, 11 in
Chapter 2].
5.2.1 Order of Reaction

The zero-order reaction is the one that the rate is independent of
the concentration of reactant.
k
S   P (5.1)
The rate (v) of formation of product is
d[S] (5.2)
v =– =k
dt
Integration of Eq. 5.2 from time 0 to t gives
[S]t = – kt +[S]0 (5.3)
where[S]t=
[S] –and
t=
kt +[S]
– kt +[S]represent the concentration of S at time t and
0 0
time 0, respectively. The substrate concentration decreases linearly
with time (Fig. 5.4). The zero-order reaction is observed in the
enzyme reaction with sufficiently high substrate concentration.
Figure 5.4 The zero-order reaction. The plot of Eq. 5.3 is shown.
The first-order reaction is the one that the rate of reaction is

dependent on the concentration of reactant. In reaction 5.1, the
rate (v) of formation of product is
d[S]
– = k[S] (5.4)
dt
Integration of Eq. 5.4 from time 0 to t gives
ln[S]t = – kt + ln[S]0 (5.5)
Equation 5.5 is expressed by the exponential function
[S]t =[S]0 e – kt (5.6)

The equation shows that the concentration of S decreases

exponentially with time. Here, the useful concept is introduced,
0.693
half-life (t1/2),= the time required for reactant to reach the half of
k
the initial amount.
Substitution for [S]t =in–[S]
Eq. =5.6
– kt
kt t+[S] 0by
+[S]0/2 gives
– kt1/2
[S]0/2 = [S]0e (5.7)

Then,
ln 2 = kt1/2
0.693 (5.8)
t1/2 =
k
0.693
Thus, the half-life (t1/2)= is determined from the rate constant,
k
and inversely the rate constant is determined from the half-life.
Figure 5.5 shows the example of the first-order reaction: the
disintegration of radioisotope, P32. P32 disintegrates to S32 with the
concomitant emission of b ray (particle). The half-life of P32 is 14.3
days, and the rate constant of the decay is 0.04846 day–1.
(a) (b)
Figure 5.5 The first-order reaction. The plots of Eqs. 5.6 (a) and 5.5 (b)
are shown. The amount of P32 is an arbitrary unit. The half-life
can be determined by the dashed line as shown in (a).
The second-order reaction is,

k
S1 + S2 P (5.9)

The rate (v) of the product formation is
dx
= k[S1][S2] = k([S1 ]0 – x )([S2 ]0 – x ) (5.10)
dt
dx
= k[S1][S 2] = k([S1 ]0 –
where and
x )([S2 ]0 –are
x ) the concentrations at time 0, and x is the
dt
product concentration at time t. Integration of Eq. 5.10 from time 0
to t gives
1 [S2 ]0 ([S1 ]0 – x )
ln = kt (5.11)
[S1 ]0 –[S2 ]0 [S1 ]0 ([S2 ]0 – x )
[S2 ]0 ([S1 ]0 – x )
The plot of ln against t will give a linear line with a
[S1 ]0 ([S2 ]0 – x )
dx dx
= k[S
= k][S
[S
slope
][S
] = kof
] ([S
=kk1([S
(]0 –1 ]x0 )([S
– x )([S
2 ]0),–2 ]thus
x0 )– x )leading to obtain the second-order rate
dt dt 1 12 2
constant k.
dx dx
= k[S
=Ink][S
[S
the][S
] case
= k] ([S
= ofk1([S
]0 –1 ]x0 )([S
–= x )([S2 ]0,–
2Eq.
]x0 )–5.11
x ) does not apply; then Eq. 5.10 is
dt dt 1 12 2
dx
 k([S1 ]0 – x )2 (5.12)
dt
Integration of Eq. 5.12 gives
x
= kt (5.13)
[S1 ]0 ([S1 ]0 – x )
The plot of Eq. 5.13 yields the second order rate constant, k.
The pseudo-first-order treatment of the second-order reaction
is convenient and simple to obtain the second-order rate constant,
and applicable in various cases. In the reaction of Eq. 5.9, the
reaction rate is expressed as Eq. 5.14:
d[S1] d[S ]
– = – 2 = k[S1][S2 ] (5.14)
dt dt
dx
When the experiments were performed= under k[S1][Sthe k([S1 ]0 – x )([S2 ]0»– x )
2] = conditions,
dx dt
= k[S1][S2] = k([S1 ].0 Then,
– x )([Sthe
2 ]0 –concentration
x) of S2 is assumed to be constant during
dt
the reaction,
dx
= k[S1][S2] = k([S
1 ]0 C– =x )([S
k 2 ] 0 – x ) (5.15)
dt
which then yields
d[S1]
– = C [S1] (5.16)
dt
This equation means the first-order reaction; so we can
determine the rate constant, C, at a given concentration of S2.
Therefore, a set of C can be obtained at various concentrations of S2.
A plot of Eq. 5.15 yields the second-order rate constant k (Fig. 5.6).
More complex reactions are given in reference [6].
Figure 5.6 Determination of the second-order rate constant by the

pseudo-first-order treatment. Plot of Eq. 5.15 is shown. The
slope of the plot gives the second-order rate constant k.
5.2.2 Practical Methods to Determine the First-Order

Rate Constant
The previous section described that even the second-order reaction
can be analyzed by the pseudo-first-order assumption. Therefore,
the method to determine the rate constant of the first-order
reaction is important and valuable for the studies on the reaction
mechanism of enzyme. Most of the commercially available stopped-
flow apparatus has its own software to fit the data for the first-order
reaction and higher-order of reactions, and show the rate constant
instantaneously.
Here, the method to determine the rate constant of the first-
order reaction is presented. The above section showed that
0.693
the first-order rate constant (k) and the half-life (t1/2)= have the
k
following relation:
0.693
t1/2 =
k
Thus, it seems easy to determine the rate constant from the half-life.
However, it is not so simple as expected. As for the reaction,
k
S  P
We could not always determine the concentration of S (or

P) directly, rather we observe the physical properties such as
absorbance, fluorescence, and so on, and relate these to the
concentration of S (or P). In these cases, the initial and final values
of these physical properties are not always easy to determine. When
it happens, we could not determine correctly the rate constant
(Fig. 5.7). When the final level of absorbance is “a,” the half-life is
0.693 0.693
t1/2(a),
= and when the final level is “b,” the half-life is t1/2(b).= As
k 0.693 0.693 k
shown in Fig. 5.7, t1/2(a) = < t1/2(b).
= Thus, the difference in the
k k
final level results in the erroneous determination of rate constant.
Figure 5.7 The determination of the first-order rate constant from the
half-life (t1/2). The absorbance follows a first-order fashion.
The final absorbances are assumed to be two cases, a and b.
Guggenheim (1926) introduced the method, known as the

Guggenheim plot, to determine the rate constant in the case that the
final level is not clear [6]. The readings of the physical properties,
such as absorbance, at times t1, t2, t3, … and t1 + D, t2 + D, t3 + D … are
References 85
l1, l2, l3, … and l1, l2, l3, …, respectively, where D is an increase of
the constant time. Then we have
ln(lt – lt ) = –kt + constant (5.17)
The pot ln(lt – lt ) vs.

= –kt + constant
t yields a linear line. Thus, the first-order
rate constant is determined from the slope.
Problems
1. In the Michaelis–Menten mechanism, the rate of reaction
is zero-order and first-order reactions at high and low
concentrations of substrate, respectively. Confirm these.
2. Derive Eq. 5.17.
References
1. Hartridge, H., and Roughton, F. J. W. (1923). A method of measuring

the velocity of very rapid chemical reactions. Proc. R. Soc. Lond. A, 104,
376–394.
2. Chance, B. (2004). The stopped-flow method and chemical
intermediates in enzyme reactions—a personal essay Photosynthesis
Res., 80, 387–400.
3. Bray, R. C. (1961). Sudden freezing as a technique for the study of
rapid reactions. Biochem. J., 81, 189–195.
4. Suzuki, H., and Ogura, Y. (1970). The kinetic behavior of the FMN
and protoheme moieties of yeast L(+)-lactate dehydrogenase
(cytochrome b2). J. Biochem., 67, 277–289.
5. Fersht A. R., and Jakes, R. (1975). Demonstration of two reaction
pathways for the aminoacylation of tRNA. Application of the pulsed,
quenched flow technique. Biochemistry, 14, 3350–3356.
6. Frost, A. A., and Pearson, R. G (1961). Kinetics and Mechanism, 2nd ed.
John Wiley & Sons, Inc. New York and London.
Chapter 6
Structure of Protein
The previous chapters described the kinetic treatments of enzyme.

We learned how enzyme efficiently catalyzes the chemical
reaction. Before 1980s, we had known from the dictionary and
the textbook that the enzyme is composed of protein. However,
the concept was denied when it was found that some RNAs have
catalytic properties. Even though some RNAs are catalytic, most
enzymes are composed of protein. From this chapter, we learn the
way to understand the catalysis on the basis of the structure of
enzyme.
6.1 Amino Acids

Amino acids are organic compounds. So, the “acid” means the
organic acid, “carboxylic acid.” The amino nitrogen and carboxyl
carbon are bound to the same carbon atom (Fig. 6.1). The structure
shows that the carbon atom binds with four groups, amino and
carboxyl groups, hydrogen atom, and R group. Thus, the central
carbon binds with four different groups. Generally, the carbon
atom bound to four different groups is called asymmetric or chiral
carbon, and the carbon atom is in the center of tetrahedron, and
four groups at its vertices. This leads to two tetrahedrons: the
stereoisomers (they are not overlapped each other) (Fig. 6.2A).

Haruo Suzuki
www.panstanford.com
88 Structure of Protein
Figure 6.1 Amino acid. Four groups are bound to the central carbon
atom. R represents the side chain.
Figure 6.2 (A) Stereo-isomer. Four groups (a, b, d, and e) are bound to the
tetrahedral carbon atom. The right configuration is a mirror
image of the left. (B) Fisher projection. From the reader, the
carboxyl group is on the top, and the side chain on the bottom,
these groups on the vertical line are pointing back of the plane
of page; The hydrogen and amino group on the horizontal line
are pointing toward the reader. As the amino group is on the
left side in the figure, the amino acid is named l-isomer.
J. van’t Hoff and J. A. LeBel (1874) reached this conclusion

independently. As described above, two isomers appear when a
molecule has one asymmetric carbon atom. This does not happen
when the central carbon and four groups are on the same plane,
since these are overlapped each other. Therefore, various methods
were proposed to present these isomers on a plane. The Fisher
projection is convenient as shown in Fig. 6.2B. The projection
rule: the groups on the vertical line are going back behind the
plane of the page, and the groups on the horizontal line are coming
out the plane of the page. The carboxyl group is on the top, and the
side chain is on the bottom [1]. If the amino group is pointing left
(or right), the amino acid is l-isomer (d-isomer). Thus, the amino
acid shown in Fig. 6.1 is l-amino acid, and the corresponding
isomer is d-amino acid. l- and d-mean left and right, respectively,
Amino Acids 89
and derived from Latin, laevus and dexter, respectively. l- and

d-Amino acids are optically active, and rotate the plane of the
polarized light. A molecule that rotates clockwise the plane of the
polarized light is dextrorotatory, and expressed “d” or “+,” and a
molecule that rotates counter-clockwise the plane is levorotatory
and expressed “l” or “–.” It had long been questioned about the
absolute configuration of d- and l-isomers. Before the absolute
configuration was determined, it was assumed that dextrorotatory
glyceraldehyde has d-configuration, and levorotatory glyceraldehyde
has l-configuration. The configuration of the other molecule
was determined from the relationship with the configuration
of glyceraldehyde. Bijovoet M. (1951) determined the absolute
configuration of sodium rubidium tartrate from the X-ray analysis.
As a result, fortunately, it was shown that d- and l-configurations
of glyceraldehyde are correct [2]. A more general notation of the
configuration of a molecule containing asymmetric carbon is RS
notation (Cahn–Ingold–Prelog notation) [1]. At first, determine
the preference of four atoms bound to the asymmetric carbon.
Figure 6.3 R, S notation of a molecule containing asymmetric carbon.

l-Ala as an example. Four atoms bound to the central carbon
are arranged in order of decreasing atomic number. When the
same atoms are appeared (this case is carbon), order according
to the atomic number of the atom (oxygen and hydrogen in this
case) bound to the atom. Thus, the preference: COOH > CH3 in
this case. Then, view from the side remote from the lowest
preference. Tracing 1 to 2 to 3 is moving counterclockwise
in the figure; thus l-Ala is assigned to be S configuration.
When tracing is moving clockwise, the central carbon is R
configuration.
The atoms are arranged in order of decreasing atomic number.

When atoms with the same atomic number appeared, compare the
atomic number of the atom bound to the initial atom. In Fig. 6.3,
two carbon atoms are bound to the asymmetric carbon. The carbon
of COOH is bound with three oxygen atoms (C=O oxygen is counted
two), but the carbon of CH3 binds with three hydrogen atoms.
Therefore, the order is COOH > CH3. Thus, the preferences of four
groups are determined. Then, view from the side remote from the
lowest preference (“H” in Fig. 6.3), and when tracing the highest
preference to the lowest rotates clockwise, then the asymmetric
carbon is “R” configuration, and when the tracing is counter-
clockwise, the carbon is “S” configuration.
Table 6.1 shows 20 proteinogenic amino acids. Only one
amino acid, Gly, has no asymmetric carbon. The 19 amino acids in
proteins are l-configuration, but “L” is not always written, since
these amino acids are mostly “L” isomer in protein. The properties
of amino acid are dependent on the nature of its side chain. In
addition to the 20 amino acids, two proteinogenic amino acids
were found. The 21st amino acid is selenocysteine (three-letter
code: Sec; one-letter code: U), in which the sulfur (S) atom in
Cys is replaced by the atom Se. Sec residue was first found in
glutathione peroxidase and coded by the opal stop codon, UGA
[3]. A number of enzymes containing Sec residue were found in
various organisms. Later, pyrrolysine (Pyl, O) was found as the
22nd amino acid residues, which is coded by the amber stop
codon, UAG [4]. These amino acids have their own tRNA and are
cotranslationally incorporated into protein molecules.
Table 6.1 Proteinogenic amino acid
Amino acid Structure of side chain (–R) pKa at 25°C

Glycine, Gly, G –H 2.35, 9.78
Alanine, Ala, A –CH3 2.35, 9.87
Valine, Val, V –CH(CH3)2 2.29, 9.74
Leucine, Leu, L –CH2CH(CH3)2 2.33, 9.74
Isoleucine, Ile, I –CH(CH3)CH2CH3 2.32, 9.76
Methionine, Met, M –CH2CH2SCH3 2.13, 9.28
Phenylalanine, Phe, F 2.20, 9.31
Amino Acids 91
Tryptophan, Trp, W 2.46, 9.41
Serine, Ser, S –CH2OH 2.19, 9.21

Threonine, Thr, T –CH(OH)CH3 2.09, 9.10
Cysteine, Cys, C –CH2SH 1.92, 10.70; 8.37
Asparagine, Asn, N –CH2CONH2 2.14, 8.72
Glutamine, Gln, Q –CH2CH2CONH2 2.17, 9.13
Tyrosine, Tyr, Y 2.20, 9.21; 10.46
Aspartic acid, Asp, D –CH2COO– 1.99, 9.90; 3.90

Glutamic acid, Glu, E –CH2CH2COO– 2.10, 9.47; 4.07
Histidine, His, H 1.80, 9.33; 6.04
Lysine, Lys, K –(CH2)4NH+3 2.16, 9.06; 10.54

Arginine, Arg, R 1.82, 8.99; 12.48
Prolinea, Pro, P –CH2CH2CH2– 1.95, 10.64

Selenocysteineb, –CH2SeH
Sec, U
Pyrrolysinec, Pyl, O
Note: Amino acids are shown, full name, three-letter code, one-letter code. The pKa
values from Dawson et al., Data for Biochemical Research, 3rd ed., Clarendon Press,
Oxford [5], and those of carboxyl and amino groups, and of side chains are shown.
aOne carbon of the side chain is bound to a-carbon, and the other carbon is bound to
the amino nitrogen.
bThe 21st amino acid.
cThe 22nd amino acid.
6.2 Polypeptide and Protein

An amino acid binds with another amino acid by the dehydration
reaction to form a dipeptide, which is composed of amino acid
residues (Fig. 6.4). The bond formed is called peptide bond. The
region surrounded by a net is called an amino acid residue, which
means the remaining part of an amino acid after removing atoms
of water molecule: hydrogen atom, a hydroxy group, or both
(Fig. 6.4b). Peptides composed of several amino acid residues are
called an oligopeptide, and peptides with more than 10 amino
acid residues called a polypeptide. A polypeptide with more than
100 amino acids is called a protein. However, a polypeptide with
shorter than 100 amino acids, such as insulin, is also called a protein.
Therefore, these namings are not rigid. The polypeptide chain
is represented like Fig. 6.4b. The N-terminal amino acid residue
is written at the left side, and the C-terminal amino acid residue is
written at the right. The residues are numbered by starting from
the N-terminal. The sequence of amino acids is called primary
structure.
(a)
(b)
Figure 6.4 (a) Formation of dipeptide by dehydration. (b) Primary

structure of polypeptide. A region in the net is called an amino
acid residue.
6.3 Analysis of Primary Structure

The primary structure of protein is the starting point to understand
the enzyme function from the structural basis. Sanger first
Analysis of Primary Structure 93
determined the primary structure of protein, insulin by his DNP

method [6]. He showed that protein is composed of amino acids and
opened the field of protein chemistry.
6.3.1 Protein Chemical Methods

Sanger developed the so-called DNP method [6]. After modification
of the N-terminal amino acid residue of a polypeptide with 1-fluoro-
2,4-dinitrobenzene, the dinitrophenylated (DNP) polypeptide was
hydrolyzed with HCl or protease under various conditions. Then,
the DNP-amino acid of the hydrolysates was identified. Repeating
these procedures, he succeeded in determining the primary
structure of bovine insulin.
Edman invented an elegant method to sequence a polypeptide
[7], now called the Edman degradation (Fig. 6.5), which is named
after him. Briefly, phenylisothiocyanate (PITC) reacts with an
amino group of the N-terminal amino acid residue of a polypeptide
under alkaline conditions to form a phenylthiocarbamylated
(PTC) polypeptide. Acidification of the PTC-polypeptide produces
the thiazoline derivative of the N-terminal amino acid residue,
and the resulting polypeptide loses one N-terminal amino acid
residue. After the extraction of the derivative from the reaction
mixture, the derivative was treated with acid to form the stable
phenylthiohydantoin (PTH-) derivative. The analysis of the
PTH-amino acid gives the N-terminal amino acid residue. By
repeating the above procedures for the polypepide deleted one
N-terminal amino acid residue, the second amino acid residue will
be given. By these procedures, we can get the amino acid sequence
of a given polypeptide. Ideally a polypeptide with an infinite
length could be determined by the method. However, the efficiency
of the reactions and the yields of the reaction products are not
100%. At best, 30 to 40 residues length may be the length to be
determined with confidence.
Edman degradation is not applicable for the proteins containing
the modified N-terminal groups, such as formylated or acetylated
N-terminal residues. Care must be taken for the analyses of proteins
containing disulfide bonds and sulfhydryl groups. Usually disulfide
bond is reduced with b-mercaptoethanol, and sulfhydryl groups
appeared are modified with monoiodoacetate to change the stable
carboxymethylated peptide, and used for the sequence analysis.

Polypeptides larger than 30 to 40 amino acid residues are not
sequenced at once. These are first cleaved into smaller fragments
by various methods (Table 6.2), then the sequences of the fragments
are determined.
Figure 6.5 Edman degradation.
The following example shows how these methods of cleavage

works (Fig. 6.6). The amino acid sequence of the polypeptide sample
is shown by one-letter code:
MMLTECPNCGPRNEFKYGGEAHVAYPEDPNALS
The polypeptide was digested with trypsin, and V8 protease.

The peptides in the digests were purified and analyzed. Then the
following data were obtained.
(1) Sequences of peptides after tryptic digestion
T1: MMLTECPNCGPR
T2: NEFK
T3: YGGEAHVAYPEDPNALS
(2) Sequences of peptides after V8 protease digestion
V1: MMLTE
V2: CPNCGPRNE
V3: FKYGGE
V4: AHVAYPE
V5: DPNALS
Sequences obtained from the analyses of peptides by one
method of cleavage (for example, tryptic peptides (T1, T2, T3)) do
not show the order of these peptides. However, the sequence of V2
overlaps with the C-terminal sequence of T1 and with the N-terminal
sequence of T2, thus the peptide T1 is connecting with T2. Similar
treatment leads to give the original sequence.
Table 6.2 Cleavage of peptide bond of protein
Methods Site of cleavage Residues related

BrCN C-terminal side of Rn Rn = Met
BrCN, anhydrous C-terminal side of Rn Rn = Met, Trp
Trypsin C-terminal side of Rn Rn = Lys, Arg, AE-Cysa
Rn + 1 ≠ Pro
Chymotrypsin C-terminal side of Rn Rn = Phe, Trp, Tyr
Rn + 1 ≠ Pro
Thermolysin N-terminal side of Rn Rn = Ile, Leu, Met, Phe, Val
Rn – 1 ≠ Pro
Pepsin N-terminal side of Rn Rn = Leu, Phe, Tyr, Trp
Rn – 1 ≠ Pro
V8 protease C-terminal side of Rn Rn = Glu, Asp
Note: Rn, the n-th amino acid residue.

aAminoethylated cysteine.
At present, it is not usual to determine the whole sequence

of protein by the chemical method described above. Rather, the
sequence analysis of the gene for protein is now popular, and
described in the next section.
Figure 6.6 Overlapping method. Peptide sample described in the text

was treated with trypsin and V8 protease, and peptides were
purified and sequenced. The sequence of V2 has the common
sequence in T1 and T2 peptides, and the V3 sequence has the
common sequence in T2 and T3 peptides.
6.3.2 cDNA Sequencing: Dideoxy Method

Proteins are translated from the respective messenger RNAs by the
protein synthesis system in the cell. mRNA contains the sequence
information for each protein. Therefore, we can get the protein
sequence by analyzing the sequence of mRNA. However, mRNA
is unstable, so the sequence of DNA complementary to that of
mRNA is determined. Various methods of DNA sequencing were
reported [8].
The dideoxy method invented by Sanger F. (1977) has been used
widely [8, 9], and its principle is shown in Fig. 6.8 [9].
A single strand DNA for sequencing and an oligonucleotide
primer complementary to the 3 region of the single strand
should be prepared. In the presence of DNA polymerase and
deoxynucleoside triphosphate, the primer is extended to produce
a new strand complementary to the original DNA strand. When
one of dideoxynucleoside triphosphates, for example, ddTTP in
Fig. 6.8a, is included in the reaction mixture, the enzyme mistakenly
incorporates ddTMP into the new strand. Then, the strand is
not able to be extended further, since ddTMP lacks 3-OH (Fig.
6.7). In Fig. 6.8a, two A bases are included in the DNA strand;
thus two new strands are observed by stopping at each of the
T bases. Figure 6.8b shows the strands to be formed in the
presence of ddATP, ddGTP, or ddCTP. When [a-32P]-labeled dATP was
used, and the polyacrylamide gel electrophoresis was performed
for the reaction mixture, all possible newly formed 11 strands
were observed as the labeled bands. Experiments with isotope-
labeled compounds need the restricted room. Therefore, nowadays,
fluorescent ddNTPs with different colors are used to label the
newly polymerized strands. A sequence of the original strand is
complementary to that determined experimentally.
5
3 2
5
3 2
Figure 6.7 Structures of deoxynucleotide triphosphate (dNTP) and
dideoxynucleotide triphosphate (ddNTP). A DNA strand
of polymerase reaction elongate from the 5-end to the 3-
end, and the 3-OH group of ribose ring is essential for the
elongation of the strand. Thus the elongation stops at the point
where the ddNTP linked to the elongating 3-end.
The length of the nucleotide sequence determined is usually

500 to 1000. How can we determine the sequence with the bigger
size of a DNA strand. As in the sequencing of a long polypeptide,
we cut them randomly. We then sequence all fragments, and we
are able to construct the original DNA sequence by the
overlapping method similar to that described for the polypeptide
sequencing (Fig. 6.6).
(a)
(b)
(c)
Figure 6.8 Schematic illustration of the principle of DNA sequencing

(dideoxy method). (a) Annealing a short oligonucleotide
primer with a DNA strand to be sequenced. Then elongate
the primer using DNA polymerase in the presence of dNTP
(dATP, dTTP, dGTP, and dCTP) and dideoxy NTP(ddATP,
ddTTP, ddGTP, or ddCTP). To prevent the complete stop of
elongation, ddNTPs are usually kept lower concentrations
than that of corresponding dNTPs. (b) Polynucleotides formed
in the presence of ddGTP, ddATP, or ddCTP. (c) Polyacrylamide
gel electrophoresis pattern of the products formed in the
presence of ddGTP(G), ddATP(A), ddTTP(T), or ddCTP(C).
Shorter strands run further from the origin (top of the gel).
Confirm the sequence by reading the nucleotide residues
from the shortest band to the longest one.
Three-Dimensional Structure 99
6.4 Three-Dimensional Structure

The specific nature of enzyme is derived from its three-dimensional
(3D) structure. The 3D structure of protein is stabilized by weak
interactions. First, weak interactions are mentioned, then the
secondary, tertiary, and quaternary structures are described.
6.4.1 Weak Interactions

Here four interactions are considered. The strength of the
interactions (bonds) is usually expressed as the energy required to
break the interactions (bonds). The bond energies of the covalent
bond is around 400 kJ/mol. However, the enegies of these four
interactions (bonds) are roughly in the range of 1 to 100 kJ/mol.
Individually the interactions are weak, but become important
when they accumulate. The order of strength are electrostatic
interaction > hydrogen bond > hydrophobic interaction > van der
Waals force.
6.4.1.1 Electrostatic interaction

In the amino acids shown in Table 6.1, Arg, Lys, and His residues
charge positively, and those of Asp and Glu residues negatively in
a neutral solution. Law of Coulomb tells us that the force (Felec,
Newton, N) between two point charges is proportional to the
charges, and inversely proportional to the length r (meter, m)
between two charges, q1q(coulomb,
2 c) and
q q (c).
Felec = 2 Felec = 1 2 2
qq 4 er 4 er
Felec = 1 2 2 (6.1)
4 er
where e(c2/Nm2) represents a permittivity of a solvent of the
charged particles. When q1q2andq1q2 have different charges, then
Felec = Felec =2
these particles attract each
4 erother,
4 erbut
2 repulsive force will work
when these particles have the same charge. The energy (Eelec,
joule) required for the two point charges apart completely is a
measure of the strength of the interaction,
q1q2
E elec = (6.2)
4 e0 Dr
q qq q
= = 1 21 2
E elecE elec
where D is a dielectric constant, and equal to4e/ .Dr0 Disr a permittivity
40e
e
of a perfect vacuum, thus D has no dimension. For example, D of
the complete vacuum is 1, and that of water approximately 80. D is

a measure of a polarity.
6.4.1.2 Hydrogen bond

A low electronegative hydrogen atom bound to the high
electronegative atom (X), such as nitrogen and oxygen, becomes
electropositive. When the hydrogen atom approaches to another
electronegative atom (Y), they attract each other. The attractive
interaction is defined to be Hydrogen Bond [10]. A typical hydrogen
bond is
X – HY
where the three dots denote the hydrogen bond. X–H represents
the hydrogen bond donor, Y the acceptor. The X – HY hydrogen
bond angle tends toward 180° and should preferably be above
110° [10]. Water dimer in vapor is hydrogen bonded each other,
and the O–H bond angle deviates 6 ± 20° from the O…O axis [11].
In proteins, the angle (X–H angle against X…Y axis) is usually not
0° due to the steric hindrance.
6.4.1.3 Hydrophobic interaction

Settling the dispersion of oil in water separates oil from water. Oil
is a hydrophobic substance, which avoids water. The phenomena
are explained as follows. Oil molecules are surrounded by water
molecules, which are linked to each other by the hydrogen bond.
Around at room temperature, when oil is transferred in water,
the change of enthalpy is small, but that of entropy is large and
negative, thus the transfer of oil is energetically not favored.
Therefore, oil molecules associate with nearby oil molecules, and
hydrogen-bonded water molecules become free, increasing the
entropy of water. Like this, the interaction among hydrophobic
molecules or groups in water is called hydrophobic interaction.
In proteins, hydrophobic amino acid residues associate each other
inside the proteins by avoiding water molecules.
The interaction of a protease with a protease inhibitor is a
good example of hydrophobic interaction. Table 6.3 shows the
thermodynamic parameters associated with the binding of protease
inhibitors to proteases. DG° values were negative with various
combinations of inhibitors and proteases, meaning that the complex
formation is favorable process thermodynamically. However, DH°

values are positive, suggesting that the complex formation is not
favorable enthalpically. On the other hand, the change of entropy
is positive, suggesting that the complex formation is entropy
driven. This may be explained as follows. That is, the hydrophobic
surfaces of proteases and inhibitors at their binding sites contain
structured water molecules forming a network of hydrogen
bonds. By the complex formation of inhibitors with proteases,
these water molecules must be freed from the structured water in
the binding site, thus increasing the entropy.
Table 6.3 Thermodynamic parameters associated with the binding of

protease inhibitors to proteases
Kd DG° DH° DS° Temp

Enzyme Inhibitor M kJ mol–1 kJ mol–1 J mol–1 K–1 pH °C Ref.
Trypsin STI 1 × 10–9 –51.4 36.0 292 5.0 25 12
Trypsin OTI 3.3 × 10–8 –42.6 23.4 222 5.0 25 12
Trypsinogen BPTI 6.3 × 10–6 –29.7 18.0 160 8.0 25 13
b‐Trypsin MTI 2.22 × 10–9 –48.5 17.1 222 8.0 21 14
b‐Chymo- RTI‐III 4.2 × 10–7 –35.6 15.5 170 8.0 21 15
trypsin
Abbreviations: STI, soybean trypsin inhibitor; OTI, ovomucoid trypsin inhibitor;

BPTI, bovine pancreatic trypsin inhibitor; MTI, mustard trypsin inhibitor; RTI-III,
proteinase isoinhibitor from oil-rape seed.
6.4.1.4 van der Waals force

The van der Waals force is named after J. D. van der Waals. Boyle–
Charles’ law is valid for the ideal gas, but not always for real gas.
This is due to the fact that the molecule of real gas has its own
volume and attracts each other. To explain the nature of real gas,
van der Waals proposed the “van der Waals equation” by considering
the volume effect and attractive force.
The atoms and/or molecules approach each other, and then
the distribution of the negative charge changes and induces dipole.
The dipole of the atom or molecule attracts electrostatically with
dipoles or electrons of other atom or molecule. The force, van der
Waals force, works when the interacting atoms and molecules
approach in the range of 0.1 to 0.2 nm, and gradually becomes
weaker when they are apart each other. On the other hand, the
repulsive force works when they approach too close because
electrons surrounding the nucleus repulse. The distance between
the nucleuses inducing the highest van der Waals force is named van
der Waals radius, and determined for atoms. For example, hydrogen,
oxygen, nitrogen, and carbon are 0.12, 0.14, 0.15, and 0.17 nm,
respectively. The van der Waals force is defined by IUPAC [16].
6.4.2 Secondary Structures and Their Determination

The peptide bond in polypeptides and proteins is in the trans-
configuration, and in the following resonance.
O O-
..
C䃐䠉C䠉N䠉C䃐
C䃐䠉C䠉N+䠉C䃐 (6.3)
H H

Figure 6.9 Conformation of the backbone chain of polypeptide. Looking

from the N-terminal end (left side in this figure) to the C-
terminal, an angle of rotation is positive when the rotation
is clockwise. In this figure, when the bond N-H on A plane
is trans-configuration to the Ca-C on B plane, the angle f is
defined 0°, and the angle y is 0° when the bond Ca-N on A plane
is trans-configuration to the bond C-O in B plane. In this figure,
f = y = 180°.
The length of the peptide bond is in between the bond length

of single and double bonds. Therefore, the rotation around the
bond is forbidden, and six atoms, Ca, O, C, N, H, and Ca, are on
the same plane (Fig. 6.9). Then, in the bonds of the backbone
structure of polypeptide chain, only N–Ca and Ca–C bonds are single
bond and can rotate. The conformation of polypeptide is
dependent on the f and y angles (defined in Fig. 6.9), and the
polypeptide chain shows ordered structures when the f and y
angles are in the range of certain values along the certain length
of polypeptide. The structure is named the secondary structure.
Figure 6.10 shows typical structures, a helix, anti-parallel b pleated
sheet structures, and b turn.
Figure 6.10 Secondary structures. Dark ribbons show the main chain
conformation of bovine pancreatic trypsin (pdb code: 2qcp).
Left: a helix, residues from Y234 to S244. Center: Completely
stretched main chains are positioned anti-parallel each other
(anti-parallel b pleated sheet). The residues from Q135 to
G140 and from K156 to P161. Right: b turn, a polypeptide
changes the direction 180°. The central amide plane (dotted
square) has two conformations, I and II. The conformation of II
is that of I rotated 180°. In each case, the main chain O atom is
hydrogen-bonded to the H atom of the N-H, thus stabilizing the
structures.
6.4.2.1 a helix
The a helix is usually a right-handed helix of polypeptide composed
of l-amino acid residues. A left-handed a helix must be rarely
observed with polypeptides with l-amino acid residues if any
because of the steric hindrance. The main chain carbonyl oxygen
atom is hydrogen-bonded with the main chain imino hydrogen
atom. The hydrogen bond forms a loop between n amino acid
residues. The number of atoms forming the hydrogen bond is 3n + 4.
For one round of a helix, 3.6 amino acid residues are included,
thus 13 atoms forms one hydrogen bond. Therefore, a helix is also
called 3.613 helix. As shown in Fig. 6.10, the side chains are on the
outside of the helix. The amino acid residues, Glu, Met, Ala, and
Leu, have a tendency to be in an a helical structure. Gly residue has

the smallest side chain (H atom), so it is free to take f and y angles
of the residue. On the other hand, Pro residue is hard to be in an
a helix due to its unique structure (Fig. 6.11).
Figure 6.11 Proline cis-trans isomerization. The peptide bond between

usual amino acid residues is a trans conformation, but a cis
conformation of X-Pro bond is found in proteins.
6.4.2.2 b sheet and b turn

b pleated sheet ( b sheet) is composed of two or more b strands
that are stabilized by hydrogen bond. b strand is usually referred
to a polypeptide fully extended along the backbone. The backbones
arrayed parallel each other are referred to as parallel b sheet,
and those anti-parallel are anti-parallel b sheet. The side chains
are on the outside of the plane of the sheet. The sheet is not simple
planar, but twisted. Most of globular proteins contain a helix and
b sheet, but these are linear structures. b turn helps to change
180° the direction of chains in protein molecule. In proteins, Pro
residue appears at the R2 position of b turn to stabilize the turn of
chains, because of its unique structure (Figs. 6.10 and 6.11). In
addition to Pro residue, Asn and Gly residues appear in the b turn.
A random coil is a protein conformation not included in a helix,
b sheet, or b turn.
6.4.2.3 Determination of secondary structures

Plane-polarized light is the sum of left-handed circularly polarized
and right-handed circularly polarized light. When an observer
looks the light source, light rotates left-handed circularly (right-

handed circularly), then the light is defined to be the left-handed
(right-handed) circularly polarized light. Circular dichroism
(CD) is the difference of absorbance between the left-handed
circularly polarized and the right-handed circularly polarized light.
A peptide group, –CONH–, is bound to a chiral carbon in a protein,
then the group becomes optically active in the region of 190 to
250 nm, and the CD spectrum is different in different secondary
structures (Fig. 6.12). The usual CD apparatus outputs the molar
ellipticity (q) expressed in degrees,
q = 3300 (eL – eR ) (6.4)
where eL and eR are the absorption coefficients of the left and right
circularly polarized light. The dimension of q is deg cm2 dmol–1.
The protein must contain various amounts of a helix, b sheet, and
random coil. For the determination of each content, the spectrum
obtained by the CD apparatus is fitted by summation of fractional
multiples of reference spectrum (like Fig. 6.12) for a helix,
b sheet, and random coil. The multiples that give the best fit for
the spectrum are the contents of each secondary structure. The
CD apparatus is usually equipped with the computer-assisted
determination of the secondary structures. CD is easy and excellent
method to estimate secondary structures, but does not give the
region of the structures along the sequence of a protein. This
weak point must be overcome by using the secondary structure
prediction.
Chou and Fasman proposed the method of prediction of the
secondary structure from the primary structure [18]. This is
based on the experiments of RNAse A by C. B. Anfinsen. RNAase A
consists of 124 amino acid residues and contains 4 disulfide groups.
When these disulfide groups were reduced by 2-mercaptoethanol
in the presence of 8 M urea, the 3D structure of the enzyme
was broken. Therefore, the enzyme completely lost its activity.
However, when 2-mercaptoethanol and urea were removed from
the mixture, the denatured enzyme recovered its 100% of the
original activity. These observations mean that the information
of 3D structure of the enzyme is involved in the primary structure.
The fact leads to the idea that 3D structure of protein must
be predicted from the primary structure. Chou and Fassman
analyzed 29 structures available in those days, and determined

the tendency of the formation of secondary structures for each
amino acid residues. The details can be seen in [18]. Various
methods to predict secondary structure have been reported and
are now available on the Web. We can easily predict the secondary
structure of proteins by loading amino acid sequence of given
proteins.
Figure 6.12 CD spectra of poly l-lysine. 1: a helix. 2: b sheet. 3: random coil.

Each spectrum is from the 100% content of each secondary
structure. Reproduced from Biochem. Biophys. Res. Commun.,
23, Townend, R., Kumosinski, T. F., Timasheff, S. N., Fasman,
G. D., Davidson, B., The circular dichroism of the b structure
of poly-l-lysine, 163–169, Copyright (1966), with permission
from Elsevier.
6.5 Tertiary and Quaternary Structures

The tertiary structure is the 3D structure of single polypeptide
chain, and the quaternary structure is the 3D structure composed
of more than 2 polypeptide chains. The methods to determine
these structures are X-ray crystallography, nuclear magnetic
resonance (NMR), and electron microscopy. X-ray crystallography
is the most popular method, but needs the crystal of protein
Tertiary and Quaternary Structures 107
concerned to succeed in the construction of 3D structure. This

means the importance of the preparation of protein crystal.
Protein crystal is solid, and it was believed that the 3D structure
constructed from the X-ray crystallography is different from the
structure in solution. It is not the case at present. The following
fact supports the idea. When enzyme crystals are dipped in a
solution containing substrate analog, the binding of the analog to
the active site of enzyme was confirmed. The 3D structure obtained
by the NMR method is good to study on the protein structure in
solution, but the size of the protein is restricted to the molecular
weight lower than approximately 25,000. Electron microscopy
uses 2D crystal of proteins, which are lined in lipid bilayer. Therefore,
the method is good for the analysis of membrane proteins such
as the proton dependent ATPase as described in Chapter 1.
It is not always possible to prepare the protein samples for
the determination of 3D structure of proteins. As described in
Section 6.3.2.3, the information of 3D structure of protein is
included in the primary structure. Therefore, the software to model
the protein structure on the basis of homology of primary structure
has been developed. SWISS-MODEL may be a representative [19],
and is a fully automated protein structure homology-modeling
server, accessible via the ExPASy web server, or from the program
DeepView (Swiss Pdb-Viewer). The methods are summarized. At
first, one must find out a template structure for a target protein to
be modeled. Perform a homology search of the primary structure
of the target protein. Then, find out 3D structure (pdb file) in
the homologous proteins found. Use the pdb file obtained as
a template for the modeling of the 3D structure of the target
protein. Figure 6.13 shows an example. The amino acid sequence
of a putative Trp monooxygenase (PTMO) from Ralstonia
solanesearum is highly homologous to that of the proenzyme of
l-Phe oxidase (deaminating and decarboxylating) (proPAO). The
detail of these enzymes will be mentioned in Chapter 10. The
enzymatic properties of the activated PTMO were identified to
be that of the activated form of proPAO (PAO). As Fig. 6.13 shows,
3D structure of PTMO is well matched with that of proPAO.
Thus, PTMO is concluded to be proPAO, and its activated form is
PAO [21].
(a)
(b)
Figure 6.13 Structural modeling of a putative Trp monooxygenase (PTMO).

The model was constructed by Geno3D [20, 21] using proPAO
structure (pdb 2yr4) as the template. (a) The stereo view of
the superimposition of PTMO monomer (red) and proPAO
monomer (blue). FAD is shown in yellow spheres. N-terminal
pro-sequences are shown as red (PTMO) and blue stick
(proPAO) models. (b) The superimposition of pro-sequence
of PTMO (red stick) and of proPAO (blue stick). Adapted from
Kurosawa et al. [21].
6.6 Structural Motif and Loop

Accumulation of 3D structures of proteins confirmed the presence
of the secondary structures described in the above sections,
and unveiled so-called supersecondary structure (motif), which
is formed by connecting secondary structures by loop (or b turn).
Loop is a random structure. Several examples of motif and the
importance of loop in the enzymatic reaction will be mentioned.
6.6.1 Supersecondary Structures: Motifs

Typical structural motifs are shown in Fig. 6.14. A helix loop helix
is a structure that two a helices are linked by loop (Fig. 6.14A).
Structural Motif and Loop 109
The motif is found in a Ca2+-binding protein and a DNA-binding

protein. This motif was first found in the structure of a carp muscle
parvalbumin [22]. The parvalbumin consists of 108 amino acid
residues, and 6 a helix: A, B, C, D, E, and F from the N-terminal to
the C-terminal residues. These are composed of three helix loop
helix, A-B, C-D, and E-F. The motif C-D and E-F bind with one Ca2+,
respectively (Fig. 6.15), but A-B helix loop helix does not bind
with Ca ion due to a short length of the AB loop. The two helices
of the motif position like fingers of right hand. Helix E (C) runs
the tip to the base of the forefinger. The middle finger corresponds
to the EF (CD) Ca2+ loop. Helix F (D) runs to the end of the thumb.
Thus, this motif is also called EF hand motif. Parvalbumin
changes its conformation by binding with Ca2+ shown in Fig. 6.15.
The conformational change works as its physiological role like
calmodulin. Calmodulin is a small molecule having 4 EF hand
motifs, and regulates the activity of various enzymes, such as Ca2+-
ATPase and protein kinase [23]. For example. the plasma membrane
Ca2+-ATPase is activated by Ca2+-calmodulin. The Ca2+-ATPase
pumps Ca2+ out of the cell. Ca2+ binds with calmodulin to change
the conformation. The conformational change leads the Ca2+-
Figure 6.14 Structural motifs. A, helix loop helix. B, coiled coil. C, zinc
finger. D, hairpin b. E, Greek key. F, bab. Nt, Ct represent the N
and C terminal sides of polypeptide, respectively. In C, C and H
represent Cys and His residues, respectively.
calmodulin to bind to Ca2+-ATPase. The Ca2+-calmodulin-bound

ATPase pumps Ca2+ from cytoplasm to the outside of the cell until
the concentration of Ca2+ becomes low enough (10–7 to 10–8 M).
Low Ca2+ concentration releases Ca2+ from the Ca2+-calmodulin
complex, thus stops the Ca2+ pump.
Figure 6.15 The conformational change of rat a parvalbumin by binding

with Ca2+. Only main chain conformation is shown. Helices
A–B (black), C–D (magenta), and E–F (orange). Following
Kretsinger and Nockolds [21], the EF region is symbolized by
right hand. Parvalbumin structures are based on the data: pdb
codes, 2jww (Ca2+ free form), and 1rwy (Ca2+ bound form).
A representative coiled coil is a structure consisting of 2a

helices wrapped around each other to form a left-handed helix
(Fig. 6.14B). There are coiled coil consisting of three to five helices,
and antiparallel helices. Amino acid residues in the interacting
region are usually hydrophobic, appearing every seventh positions
over several helical turns. The leucine zipper motif is the example
of the coiled coil. A coiled coil regulates various function of DNA
by binding with DNA molecule. Figure 6.16 shows how a coiled
coil binds with DNA.
A zinc finger was first found in a Xenopus transcriptional
factor, and the name derives from the discovery that the zinc-
binding domains are used to grip the DNA [24]. The term is
now used to identify any compact domain stabilized by zinc ion.
Several different types of zinc ligands are known. The most well
studied is probably a C2H2 type shown in Figs. 6.14C and 6.16.
Others are C2HC, C3H, and C4, which ligate one zinc ion. A zinc 2 C6
type is also known. Here, C and H represent cysteine and histidine
residues, respectively.
Structural Motif and Loop 111
Figure 6.16 Examples of a coiled coil, zinc finger, hairpin b, and Greek
key. Structures are shown by ribbon. The coiled coil and zinc
finger motifs are bound to groove of a DNA double helix. Pdb
code:1ysa (coiled coil), 1zaa (zinc finger), 1k6u (hairpin b),
and 4gcr (Greek key). A double Greek key is shown.
A hairpin b ( b hairpin or bb) is a structure that two b strands

are connected antiparallel with a short loop (Fig. 6.14D). Figure
6.16 shows the hairpin b region of bovine pancreatic trypsin
inhibitor.
A Greek key is composed of four b strands, which are positioned
antiparallel (Fig. 6.14E). An eye lens protein, gamma-B crystalline,
contains two domains composing a pair of Greek key motif
(Fig. 6.16). A Greek key is also observed in the Fab region of
immunoglobulin G.
A bab is a motif that parallel b strands are connected with a
loop and an a helix (Fig. 6.14F). That is, the secondary structures
are connecting b strand-loop-a helix-loop-b strand from the
N-terminal side. The motif is named Rossman fold after M.
Rossman [25], who first pointed out that the motif are present in
nucleotide-binding proteins. For example, the ADP moiety of FAD in
l-Phe oxidase is bound to the first loop of the motif (Fig. 6.17A).
This region has the characteristic sequence Gly-X-Gly-X-X-Gly,
which composes the binding site of the nucleotide ligands.
A loop is a structure that links secondary structures as
described above. Besides a linker, a loop has functional roles of
proteins, such as enzyme activities, and ligand binding in antibodies
and receptors. Triose phosphate isomerase (TIM) is one of the
well-characterized and almost perfectly evolved enzyme [26]. TIM
is the key enzyme in glycolysis, and catalyzes the interconversion
of dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde
3-phosphate (GAP). The kcat/Km value of TIM is 3.7 × 108 M–1s–1 for
GAP and >107 M–1s–1 for DHAP as substrate. These values are very
close to the diffusion limit, thus TIM is “almost perfectly evolved”
enzyme. A loop has an important role for the enzyme function.
The structure of TIM is consisting of the bab motif (Fig. 6.17B,C).
The residues Trp168 to Thr177 are composing a flexible loop,
which is covering substrate in the active site. Knowles’ group
showed that the removal of 4 residues (from residue 170: Ile-Gly-
Thr-Gly) gave approximately 105 times lower activity of the wild-
type enzyme. Probably, the loop must prevent a cis-endiol
intermediate from decomposing to methyglyoxal and phosphate.
Figure 6.17 A, bab motif. A carbonyl group of A66 in l-Phe oxidase (pdb
code: 3ay1) interacts with the phosphate group of FAD (only
ADP region is shown). B, C. Triosephosphate isomerase
(TIM) barrel (pdb code:7tim). A substrate analogue,
phosphoglycohydroxamate (cyan spheres), is covered with
loop (pink). Red spheres are the active site residues. Eight
a helices and eight b strands form a barrel-like structure,
thus called TIM barrel.
Lactate dehydrogenase (LDH) catalyzes the dehydrogenation

of lactate to form pyruvate, with concomitant production of
NADH from NAD+. The 3D structures of LDH revealed that a mobile
loop (residues 98 to 110) covers the active site when substrate
binds to the site. Waldman et al. (1988) determined the rate of
closure of the loop, and proposed that the rate limits the overall
reaction rate [27]. That is, they introduced Trp residues at the
residue 106 in the loop, and all the Trp residues were mutated to
Tyr. Then Trp fluorescence of LDH comes from the loop’s Trp
residue. They observed the time-dependent fluorescence
decrease after mixing the LDH-NADH complex with the pyruvate
References 113
analog, oxamate (H2N-COCOOH). The time-course of the

fluorescence decrease agreed well with that of decrease of NADH
after mixing the LDH-NADH mixture with pyruvate. This fact
clearly shows that the loop closure limits the rate of overall
reaction.
Problems
1. RNAase A contains four disulfide bonds. After reduction of

the bonds, we get eight cysteine residues. By oxidation of
these residues, four disulfide bonds will be formed.
Considering the disulfide shuffling, how many combinations
of disulfide bond are possible?
2. Fluorescence of Trp on the mobile loop of LDH decreases by
binding with the pyruvate analog, oxamate (Section 6.6.1).
Why is the fluorescence decreased?
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homology modelling. Bioinformatics, 22, 195–201. SWISS-MODEL
is a fully automated protein structure homology-modeling server,
accessible via the ExPASy web server, or from the program DeepView
Swiss Pdb-Viewer.
20. Combet, C., Jambon, M., Deleage, G., and Geourjon, C. (2002). Geno3D:
Automatic comparative molecular modeling of protein. Bioinformatics,
18, 213–214.
References 115
21. Kurosawa, N., Hirata, T., and Suzuki, H. (2009). Characterization of

putative tryptophan monooxygenase from Ralstonia solanasearum.
J. Biochem., 146, 23–32.
22. Kretsinger, R. H., and Nickolds, C. E. (1973). Carp muscle calcium-
binding protein II. Structure determination and description. J. Biol.
Chem., 248, 3313–3326.
23. Lupas, A. N., and Gruber, M. (2005). The structure of a-helical coiled
coils. Adv Protein Chem., 70, 37–78. (review).
24. Krishna, S. S., Majumdar, I., and Grishin, N. V. (2003). Structural
classification of zinc fingers: Survey and summary. Nucleic Acids Res.,
31, 532–440.
25. Rao, S., and Rossmann, M. (1973). Comparison of super-secondary
structures in proteins. J. Mol. Biol., 76, 241–256.
26. Knowles, J. R. (1991). Enzyme catalysis: Not different, just better.
Nature, 350, 121–124 (review).
27. Waldman, A. D. B., Hart, K. W., Clarke, A. R., Wigley, D. B., Barstow, D. A.,
Atkinson, T., Chia, W. N., Holbrook, J. J. (1988). The use of a genetically
engineered tryptophan to identify the movement of a domain of B.
stearothermophilus lactate dehydrogenase with the process which
limits the steady-state turnover of the enzyme. Biochem. Biophys. Res.
Commun., 150, 752–759.
Chapter 7
Active Site Structure
Chapter 6 described the overall structure of protein. This chapter

describes structures of the regions related to enzyme functions. The
first half of the chapter deals with cofactors, and the rest discusses
the methods to study the active site structure.
7.1 Active Site and Active Center

In the previous chapters, the term active site was used without
definition. The active site means the region (area or place) of enzyme
protein where substrate binds and is transformed to product. For
the same meaning, the active center has been used. The “center”
means “point.” However, accumulation of 3D structures of enzyme
has shown that the region of enzyme related to the function is broad
in space. Therefore, it seems better to use the term “active site.”
7.2 Cofactor, Coenzyme, Prosthetic Group

The active site of enzyme is usually composed of several amino
acid residues. However, various enzymes also require metal ions,
and/or organic groups for the activities. The terms used to denote
these groups seem to be slightly different from one book to another.
Here, the following usage is applied in this book. A cofactor means

Haruo Suzuki
www.panstanford.com
118 Active Site Structure
substances required for the activity of enzyme. The cofactor includes

metal ions, coenzyme, and prosthetic groups [1].
Coenzyme binds with the enzyme protein reversibly, and acts
like substrate. Prosthetic group is an organic compound, binds
strongly with enzyme, and is usually present as a protein-bound
form. The cofactor-bound enzyme (protein) is called holoenzyme
(holoprotein), and the cofactor-unbound enzyme (protein) is
apoenzyme (apoprotein). Table 7.1 shows main cofactors. Several
examples will be shown in the following sections.
Table 7.1 Main cofactors
Cofactors Main functions

ATP Transfer of phosphate and
nucleotide
Nicotinamide adenine dinucleotide Two electron transfer of
(NAD+); nicotinamide adenine oxidation-reduction reaction
dinucleotide phosphate (NADP+)
Coenzyme A (CoA or CoA-SH) Acyl group transfer
Folic acid Transfer of single-carbon
Flavin mononucleotide (FMN); flavin One or two electron transfer of
adenine dinucleotide phosphate (FAD) oxidation–reduction reaction
Heme One electron transfer of oxidation-
reduction reaction
Thiamine pyrophosphate (TPP) C–C bond cleavage
Pyridoxal phosphate (PLP) Group transfer of amino acid
Biotin Transfer of carboxyl group,
carboxylation
Adenocylcobalamin; methylcobalamin Rearrangements, methyl group
transfer
Lipoamide Acyl group transfer
Note: Abbreviation of cofactor is shown in the parentheses.
7.2.1 NAD+ (Nicotinamide Adenine Dinucleotide) and

NADP+ (Nicotinamide Adenine Dinucleotide
Phosphate)
A. Harden and W. Young (1905) studied the alcoholic fermentation.
The fermentation requires the presence of both a heat-labile
Cofactor, Coenzyme, Prosthetic Group 119
component in the residue (zymase) and a low molecular weight,

heat-stable component in the filtrate (coferment, coenzyme).
Chemical nature of coenzyme was not known in those days, but
it was later shown that the coenzyme contains NAD+ [2, 3]. NAD+
and NADP+ act in the oxidation-reduction reactions (Fig. 7.1). For
example, l-lactate dehydrogenase catalyzes the following reaction:
CH3CH(OH)COO– + NAD+ 

 CH3COCOO– + NADH + H+ (7.1)

The structural and mechanistic studies revealed that hydride ion
is removed from a hydrogen atom of lactate to form NADH. As
Eq. 7.1 shows, NAD+ (NADH) acts like substrate in various enzymatic
reactions.
Figure 7.1 Oxidized and reduced forms of NAD+ (NADP+). R1 is H for

NAD+ and PO2– + + +
3 for NADP . The pyridine ring of NAD (NADP )
receives hydride ion to be reduced to NADH (NADPH).
7.2.2 Coenzyme A (CoA, CoA-SH)

CoA is involved in a metabolic pathway of pyruvate produced by
glycolysis, and in various steps of lipid metabolism. The functional
group of CoA is terminal sulfhydryl group (Fig. 7.2). For example, a
pyruvate dehydrogenase complex is a multienzyme complex, which
is composed of various enzymes and cofactors. The overall reaction
catalyzed by the pyruvate dehydrogenase complex is,
CH3COCOO + HS–CoA + NAD+ CH3CO–S–CoA + NADH + CO2 + H+ (7.2)
The bond between the carbonyl carbon and sulfur in acetyl-CoA

is the high energy bond. The energy level is the value similar to
the energy of the ATP hydrolysis (–32 kJ/mol). The phosphoryl

group of the phosphopantetheine moiety of CoA forms an ester
bond with a hydroxyl group of Ser residue of acyl carrier protein
(ACP), and composes the prosthetic group of ACP. ACP is involved
in lipid metabolism.
Figure 7.2 Coenzyme A and phosphopantetheine prosthetic group.
7.2.3 Flavin Mononucleotide (FMN), Flavin Adenine

Dinucleotide (FAD)
These cofactors are derived from vitamin B2 (riboflavin), and
are bound to proteins noncovalently or covalently. The enzyme
containing these cofactors catalyzes dehydrogenation of substrate,
and is called flavo-enzyme, or flavin enzyme [4]. The flavin enzyme
acts as an electron acceptor from the reduced coenzyme (for
example, NADH and reduced lipoamide), and acts to reform the
reduced coenzyme from the oxidized coenzyme (Fig. 7.3). Oxidation
Figure 7.3 Oxidation-reduction of FAD and FMN. Isoalloxazine ring is

indicated by a square dashed line.
of amino acids, carbohydrate, and fatty acids is also catalyzed by

flavin enzymes. When the electron acceptor is oxygen, the enzyme
is called oxidase as shown in Eq. 7.3, and when the acceptor is not
oxygen, the enzyme is called dehydrogenase.
R–C(NH3+ )COO– +O2 + H2O R–CO–COO– + NH+4 + H2O2. (7.3)

7.2.4 Heme
Heme is a compound that has a structure shown in Fig. 7.4. Four
pyrrole rings are linked by methine to form porphyrin ring. The
center of the ring contains iron, and the difference in the side chains
composes different hemes, such as cytochrome a, b, c, and so on. The
structure minus iron in Fig. 7.4 is named protoporphyrin IX (also
called heme b). Heme is the red component in oxygen transport
proteins, hemoglobin and myoglobin. The central ferrous iron of
these proteins binds with oxygen. Heme is a prosthetic group of the
oxidation-reduction enzymes, such as peroxidases, cyclooxygenase,
cytochrome b2 (l-lactate dehydrogenase), nitric oxide synthase,
and cytochrome P450. In addition, cytochromes have a role of one
electron transfer in mitochondrial electron transfer system.
Figure 7.4 The structure of heme. A region of the dashed circle indicates
porphyrin ring with iron in the center.
7.2.5 Pyridoxal Phosphate (PLP)

Pyridoxal phosphate (PLP) has a key role in amino acid metabolism
as a cofactor of enzyme, and is derived from vitamin B6 (Fig. 7.5).
The PLP-dependent enzymes catalyzes the transformation of amino

acids, such as transamination, decarboxylation, racemization, and
synthesis. Pyridoxal phosphate binds covalently with the ε amino
group of the enzyme protein as a Schiff base. The Schiff base is the
double bond formed by the reaction of the primary amine with a
carbonyl group (aldehyde and ketone). When an amino acid binds
with the PLP-enzyme complex, a Schiff base with the enzyme is
exchanged with the amino group of the amino acid (Fig. 7.6). After
the formation of PLP-amino acid complex, the bound amino acid
changes to products differently from one enzyme to another. In
other words, the active site structure around the PLP-amino acid
complex determines which way amino acids take [5].
Figure 7.5 Vitamin B6 group.
Figure 7.6 PLP forms a Schiff base with the side chain of Lys residue of
enzyme. Amino group of substrate amino acid is exchanged
with the e amino group of the Lys residue.
7.2.6 Folate
Folate is composed of pterin, p-amino benzoate, and glutamate
residue (Fig. 7.7). 5,6,7,8-tetrahydrofolate (THF) works as a cofactor
of the C1 transfer reactions, such as formylation of Met-tRNA,
syntheses of Gly, Ser, Met, and pyrimidine and purine nucleotides
[6]. The Glu residue of THF presents as tri- to hexamer, and has a
role to bind with enzyme protein. THF is synthesized by two steps
reduction of folate by way of 7,8-dihydrofolate. The C1 donors are
synthesized enzymatically from THF. 5,6,7,8-Tetrahydrobiopterin
is a cofactor of monooxygenase, such as Phe hydroxylase and Tyr
hydroxylase.
Figure 7.7 Structure of folate derivatives.
7.2.7 Thiamine Pyrophosphate

Thiamine is the first vitamin found. The name vitamin is derived from
“vital amine.” Thiamine pyrophosphate (TPP) or differently called
thiamine diphosphate (TDP) is a cofactor of enzymes catalyzing C–C
bond-forming and bond-breaking reactions. Pyruvate decarboxylase

and transketolase are the examples. The pyruvate decarboxylase
catalyzes the following reaction:
CH3COCOO–  CH3CHO + CO2 (7.4)
The reaction site of TPP is the C2 carbon of thiazolium ring (Fig.

7.8) [7]. The first step of the enzyme catalyzed reaction is the
formation of the C2 carbanion of TPP. Breslow (1957) observed that
the C2 proton of thiamine exchanges with solvent deuterium with a
half-life of about 20 min at 28°C. However, Washbaugh and Jencks
(1988) reported very high pKa of 17.7 of the C–H bond dissociation.
The pKa value means as follows. Under neutral conditions where
enzymes are usually active, the concentration of the C2-ionized
form (carbanion, ylide) is estimated to be very low to proceed the
catalysis. How enzyme overcomes the problem. As for pyruvate
decarboxylase and transketolase, the uncharged side chain carboxyl
of the Glu residue is hydrogen-bonded with N1¢ of the pyrimidine
(Fig. 7.8c). A proton transfer from the carboxyl group of the Glu
residue to N1¢ leads to the proton release from the C2-H, via the
imino-amino tautomerization (N1¢ and N4¢ of pyrimidine ring) [7].
The C2 carbanion formed attacks the carbonyl carbon of substrate
(a) (c)
(b) (d)
+ H+
Figure 7.8 The activation of TPP. (a) Structure of TPP. (b) The hydrogen
atom (dashed circle in a) bound to the thiazolium C2 is released
as H+, and the C2 carbon becomes negatively charged. (c) The
active site structure of pyruvate decarboxylase (pdb: 1pyd).
The distance (2.4 Å) between the carboxy O atom of Glu51 and
N1¢ atom of TPP, and the internal distance (3.0 Å) from N4¢ to
C2 of TPP are sufficient for H+ transfer. (d) The C2 carbanion
attacks the carbonyl carbon of substrate, forming a common
intermediate in TPP-dependent enzymes.
to form a covalent intermediate. That is, the acetyl carbonyl carbon

corresponds to the carbon to be attacked in pyruvate decarboxylase
reaction (Eq. 7.4). This kind of the covalent intermediate is a common
intermediate catalyzed by TPP-dependent enzymes (Fig. 7.8d) [7].
7.2.8 Biotin
Biotin is a cofactor of enzymes that catalyze carboxylation reactions.
Biotin is covalently bound to the enzyme through an amide bond
between the carboxyl group of biotin and the e amino group
of Lys residue of enzyme (Fig. 7.9). Pyruvate carboxylase is a
representative of carboxylating enzymes [8]. The first step is an
ATP-dependent formation of the carboxybiotin, and then it reacts
with enolpyruvate to produce oxaloacetate (Fig. 7.9). Here
enolpyruvate is a tautomer of pyruvate.
(a)
(b)
Figure 7.9 (a) Biotin bound to the e-amino group of the side chain of Lys
residue of enzyme. The dashed circle indicates the reaction
site of the cofactor. (b) A scheme of formation of oxaloacetate
from enolpyruvate and carbonic ion.
7.2.9 Lipoamide
Lipoamide is a cofactor that catalyzes the acyl group transfer
reaction in pyruvate dehydrogenase complex and 2-oxoglutarate
dehydrogenase complex. The cofactor covalently binds to enzyme
protein by the amide bond with the e-amino group of Lys residue
(Fig. 7.10a). In the pyruvate dehydrogenase complex, lipoamide
receives acetyl group from hydroxyethylthiamine pyrophosphate to
form acetyl-lipoamide Fig. 7.10b), which transfers the acetyl group
to CoA-SH to form acetyl-S-CoA.
(a)
(b)
Figure 7.10 Lipoamide cofactor and its function. (a) Structure of lipoamide
cofactor. (b) In pyruvate dehydrogenase complex, lipoamide
receive methyl group from hydroxyethylthiaminepyrophos-
phate to produce acetyldihydrolipoamide, which transfer
acetyl group to Coenzyme A.
7.2.10 Protein-Derived Cofactors

These are derived from the post-translational modification of
protein, that is, the oxygenation of aromatic residues, covalent
cross-linking of amino acid residues, or cyclization or elongation of
internal amino acid residues [9]. The first cofactor in this category
is Topaquinone found in Cu2+-amine oxidase [10].
Search of Active Site 127
7.3 Search of Active Site

The previous section showed the importance of the cofactor in
the catalysis. Moreover, we learned the importance of amino acid
residues around cofactors. Chapter 6 described that the structure
of enzyme can be determined by the physico-chemical methods.
However, special apparatus and techniques are required for such
studies. This section describes universal and easy ways to study on
the active site: chemical modification and site-directed mutagenesis.
Care must be taken not to induce large structural changes by
these methods. The far UV CD measurements of the modified and
unmodified enzymes are usually used to test if or not structural
changes occur by these methods. The chemical modification may be
useful to primary and pilot studies on the active site of enzyme.
7.3.1 Chemical Modification

In amino acid residues in the enzyme proteins, functional groups
are amino, carboxyl, sulfhydryl, hydroxyl, imidazole, and gunidino
groups. Table 7.2 shows typical modification methods of these
groups. Each method will be briefly explained [11, 12].
7.3.1.1 Amino group

Acetic anhydride is frequently used for the modification of amino
group of protein, and reacts with amino group as Eq. 7.5. Amino group
in proteins is the amino terminal a-amino group and e-amino group
of Lys residue. The pKa of these groups in free state are roughly 8
and 9, respectively. Therefore, under the conditions below these
pH, the rate of reaction of amino group with acetic anhydride is low.
Usually modifications are performed alkaline conditions.
(7.5)

Table 7.2 Reagents for chemical modifications
Functional groups Reagents

Amino Acetic anhydride
Carboxyl Glycine ethylester, water-soluble
carbodiimide
Sulfhydryl Iodoacetate; iodoacetamide;
p-mercuribenzoate;
5,5-Dithiobis(2-nitrobenzoic acid)
(DTNB); N-ethylmaleimide (NEM)
Hydroxyl Diisopropylfluorophosphate
Tetranitromethane
Guanidino Phenylglyoxal
Imidazole Diethylpyrocarbonate
Indole 2-Hydroxy-5-nitrobenzyl bromide
7.3.1.2 Carboxyl group

The carboxylate anion of Asp and Glu residues reacts with a water-
soluble carbodiimide (WSC), then with amine to produce amide
(Eq. 7.6). The reactions are performed under acidic conditions
(pH 5). The reagents also react with sulfhydryl group of Cys residue
and phenolic hydroxy group. The extent of modification can be
tested by treating reaction products with hydroxylamine, since
hydroxylamine cleaves the ester bond to reform carboxyl group
(E-COO– to E-CONHCH2COO–), though the –N–C- bond is inserted.
(7.6)

7.3.1.3 Sulfhydryl group

For the modification of sulfhydryl group, various compounds were
synthesized.
Iodoacetamide, iodoacetate: These are highly reactive with
sulfhydryl group under neutral pH conditions (pH 6 to 8).
E-SH + ICH2CONH2  E-S-CH2CONH2 + H+ + I

E-SH + ICH2COO  E-S-CH2COO + H+ + I (7.7)
p-Mercuribenzoate: This reagent reacts with sulfhydryl groups

under acidic conditions (pH 4.5 to 5) (Eq. 7.8), and is conveniently
used to detect the extent of modification by measuring the
absorbance increase of the reaction mixture at UV region. Care
must be taken to use the reagent, since it is an organic mercury and
poisonous.
(7.8)

5,5-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman reagent): DTNB

reacts with the sulfhydryl group stoichiometrically to produce
yellow compound (thionitrobenzoate anion); thus, it is widely used
to quantitate the amount of sulfhydryl group in proteins (Eq. 7.9).
The modified enzyme will be reactivated by treating with
b-mercaptoethanol, which reduces the disulfide bond.
(7.9)
N-ethymaleimide (NEM): The reaction of NEM with sulfhydryl group

is selective under low pH (pH 5 to 7). The rate of NEM reaction
with sulfhydryl group increases with increasing pH, but NEM also
reacts with amino group at higher pH (>7).
(7.10)
7.3.1.4 Hydroxyl group

Ser, Thr, and Tyr residues contain hydroxyl group. The reagents used
are diisopropylfluorophosphate (DFP) and tetranitoromethane
(TNM).
Diisopropylfluorophosphate (DFP): DFP is the representative

organophosphate, and poisonous. Modification of Ser residue
of acetylcholine esterase with DFP (Eq. 7.11) is already
mentioned in Chapter 1. Ser residues of trypsin and horse serum
butyrylcholinesterase are reported to be modified.
(7.11)
Tetranitoromethane (TNM): TNM is a yellow toxic compound, and

reacts with Tyr residue under mild conditions (Eq. 7.12). TNM
itself is not explosive, but explosive in the presence of impurities.
The nitrated Tyr residue is stable in hot acid, and shows visible
absorption under alkaline conditions. This property is used to
monitor the extent of modification. The pKa of nitrated Tyr is
around 7, therefore more acidic than Tyr. Recently, peroxynitrite
(ONOO–) is widely used to modify Tyr residue.
(7.12)

7.3.1.5 Guanidino group
Phenylglyoxal(PGO): The guanidino group of an Arg side chain has
critical role for the binding of the negatively charged substrate.
PG reacts with guanidino group of the Arg side chain under mild
conditions (Fig. 7.11). Takahashi (1968) invented the method,
Figure 7.11 Reaction scheme of PGO with a guanidino group of Arg residue.
Binding of one and two molecules of PGO is shown [13–15].
showed that the active site Arg residues in RNAase A are modified,
and that two moles of PGO react with one mole of Arg residue
[13]. However, several cases showed that one mole of PGO reacts
with one mole of Arg residue [14, 15]. Glyoxal, methylglyoxal, and
p-hydroxylphenylglyoxal also reacts with guanidino group.
7.3.1.6 Imidazole group

Imidazole group of His residue ionizes around neutral pH.
Therefore, His residues have been shown to be critical role in
various enzymes. Diethypyrocarbonate is selective for the imidazole
group of His residue. Usually reactions are performed under pH 7.
The specific ethoxyformylation of His residue can be confirmed by
observing the recovery of the activity with the mild hydroxylamine
treatment of the modified enzyme, since the ethoxyformyl group
can be removed by the treatment. This modification increases the
absorbance around 240 nm; thus, we can estimate the extent of
reaction by monitoring the absorbance change.
(7.13)

7.3.1.7 Indole group

The indole group of Trp residue is specifically modified by
2-hydroxy 5-nitrobenzyl bromide (HNBB) under neutral conditions
(Eq. 7.14). Koshland et al. (1964) used the compound to modify
Trp residue of a-chymotrypsin. Then various enzymes were
modified by the reagent.
(7.14)

7.3.2 Site-Directed Mutagenesis

The method requires an expression system to synthesize a desired
enzyme. Here, we assume that we have an E. coli expression system
for a desired protein. The method is summarized in Fig. 7.12. At
first, design and synthesize complementary mutagenic primers,
sense and anti-sense primers. The sequences of primers must
contain the target nucleotide to be mutated, and are better to have

10 to 15 nucleotides length in both side of the target nucleotide.
Denature the plasmid containing the target site, anneal the primers,
and extend the primers using DNA polymerase, thus leading to the
formation of nicked circular strands. These procedures are run
over 10 times using a thermal cycler. The template plasmid DNAs
are methylated, but DNAs synthesized by PCR are not methylated.
The restriction endonuclease DpnI recognizes the methylated
adenine in the sequence GATC, and cleaves the phosphodiester
bond between the methylated adenine nucleotide and thymine
nucleotide. The DpnI treatments of the PCR products cleave the
template plasmid, and leave the PCR products with the mutated
sequence. Then, incubate the mixture with competent E. coli cells,
which repairs the nicks in the mutated plasmids. Thus, we can
prepare E. coli cells harboring the mutant plasmid. Here competent
E. coli cells mean those that can transport relatively small DNA such
as plasmid and phage through the cell membrane. In laboratory,
competent cells are prepared by treating the E. coli cells with Ca2+
at low temperature. Various methods for site-directed mutagenesis
have been invented. Practically a mutagenesis kit is commercially
available.
Figure 7.12 Site-directed mutagenesis using a whole plasmid.
7.3.3 Examples of Active Site Studies

There are accumulated studies on the active site of enzyme. Here,
clear-cut examples are shown.
7.3.3.1 Chemical modification of l-Phe oxidase

l-Phe oxidase (PAO) from Pseudomonas sp. P-501 catalyzes both
oxidative deamination and oxygenative decarboxylation of l-Phe,
and highly specific for l-Phe. The enzyme is composed of 2 a and
2 b subunits, and contains 2 FAD. Details on the enzyme will be
described in Chapter 10. In the reagents in Table 7.2, compounds
containing benzene ring might be a pseudo-substrate and an
active site-directed modifier of PAO. p-Hydroxymercuribenzoate
had no effect on the activity; however, phenylglyoxal (PGO) and
p-hydroxyphenylglyoxal inhibited the enzyme. 14C-Labeled PGO is
commercially available; therefore, mechanism of inhibition by PGO
was studied by Mukouyama et al. [16].
The inhibition of the enzyme by PGO can be expressed as
follows:
k
E + n PGO   E(PGO)n (7.15)
where E represents the active enzyme-unit containing one FAD,
k
and
E + n PGO   E(PGO)n the inactive form of enzyme-unit modified with n
molecules of PGO. Then, the rate of the inactivation of enzyme will
be
d[E]
v=– = k[E][PGO]n (7.16)
dt
When the concentration of PGO is sufficiently higher than that
of enzyme-unit, the treatment of the pseudo-first order reaction can
be applied as described in Chapter 5.
d[E]
v=– = kobs [E] (7.17)
dt
where
kobs = k [PGO]n (7.18)
Integration Eq. 7.17 from time 0 to t,
ln[E]t = –kobst + ln[E]0 (7.19)
The concentration of the active enzyme-unit is proportional to the

enzyme activity. Therefore, the enzyme was incubated with various
concentrations of PGO for given times, then the enzyme activity
was assayed to measure the concentration of the active enzyme.
A plot of ln(activity) vs. time gave a linear line as shown in Fig. 7.13a
(Eq. 7.19), supporting that the inactivation follows the first-
order reaction. The plot of the first-order rate constant (kobs ) of
inactivation vs. [PGO] was linear, indicating that one PGO molecule
binds with one enzyme-unit (Fig. 7.13b). To confirm this conclusion,
log kobs vs. log [PGO] was plotted (Fig. 7.13c). From Eq. 7.18,
(a) (b)
(c)
Figure 7.13 The chemical modification of l-phenylalanine oxidase with

PGO. (a), the enzyme (24 μM enzyme-unit) was preincubated
at pH 9 with PGO as shown, and a part of the mixture was
assayed for enzyme activity and plotted. (b) the first-order
rate constant (kobs) obtained from (a) was plotted against the
concentration of PGO. Adapted from Mukouyama et al. [16].
log(kobs) = log k + n log[PGO] (7.20)
Thus, the slope of the plot gives the number of PGO molecule
reacted with the enzyme-unit. Figure 7.13C shows a linear plot with
the slope of 1.03, confirming that one PGO molecule binds with one
enzyme-unit.
(a)
(b)
Figure 7.14 Sodium dodecyl sulfate gel electrophoresis of the 14C-labeled

enzyme. (a) Protein staining. (b) Autoradiography. a and
b indicate the migration position of the enzyme subunits.
M, molecular weight marker. Numbers indicate the time of
incubation (min) with the 14C-labeled PGO. Adapted from
Mukouyama et al. [16].
(a) (b)
(c)
Figure 7.15 HPLC analysis of the lysylendopeptidase digests of the

14C-labeled b subunit. (a) Absorbance change at 210 nm of
eluate. (b) and (c), the radioactivity of each fraction of the

eluate. The b subunit labeled with [14C]PGO in the absence
(b) and presence of l-Phe (c). Adapted from Mukouyama et al.
[16].
As the inactivation of enzyme with PGO is prevented by l-

Phe, the Arg residue modified by PGO must be in the substrate-
binding site. To know the structure around the PGO-modified
Arg residue, the enzyme was incubated with 10 mM [14C] PGO in

the presence and absence of 20 mM l-Phe at pH 9, and a part of
the mixture was taken and mixed with 286 mM unlabeled PGO.
The whole mixture was boiled in the denaturation buffer for sodium
dodecylsulfate gel electrophoresis. Figure 7.14b shows that the
labeling by PGO is in the b subunit and is prevented by l-Phe. To
identify the modified residue, 14C-labeled b subunit was digested
with lysylendopeptidase, and purified by high performance
liquid chromatography (HPLC) (Fig. 7.15). The labeled peptide
in the fraction (shown by arrow in Fig. 7.15) was purified, and
the amino acid sequence was analyzed. The PGO-labeled amino
acid residue is identified to be Arg35 of the b subunit. Now the
guanidino group of the residue has been identified to be bound to
the carboxylate anion of substrate amino acid by the X-ray
crystallography [17].
7.3.3.2 Site-directed mutagenesis of thermostable l-lactate

dehydrogenase
l-Lactate dehydrogenase (LDH) catalyzes as follows:
CH3COCOO– + NADH + H CH3CH(OH)COO + NAD (7.21)
This reaction is in the last step of glycolysis. Bacterial LDHs

shows allosteric property. That is, the enzyme is greatly activated
with fructose 1,6-bisphosphate (FBP) (allosteric effector). The
meaning of allosteric will be mentioned in detail in Chapter 8. FBP
is formed as the last metabolite of 6 carbon carbohydrates in the
glycolytic pathway. LDH from Thermus caldophilus GK24 loses the
allostericity by chemical modification of Arg-specific reagents
such as 1,2-cyclohexadione and 2,3-butanedione. Matsuzawa et al.
[18] modified the enzyme by 2,3-butanedione, and identified
the labeled amino acid residue to be Arg-173 in the sequence of
Phe-Arg173-Ala-Leu-Leu-Ala-Glu-His-Leu-Arg. Based on the results,
the residue was mutated to the Gln residue. The mutant enzyme
was prepared and purified. The activity of wild-type enzyme was
stimulated by FBP, but that of the mutant enzyme was the same
with and without added FBP (Fig. 7.16). That is, the allosteric
nature of the wild-type enzyme was lost by mutation of Arg-173
to Gln. The results indicate that the guanidino group of Arg-173
References 137
interacts with FBP, resulting in the activation of LDH. The idea has
been supported by the 3D structure of the enzyme-FBP complex
(pdb: 3vph), showing that FBP is bound the amino acid residues
including H188 at the interface of two subunits.
Figure 7.16 Change of thermostable LDH from an allosteric to a non-

allosteric form by mutation of Arg-173 to Gln [18]. Adapted
from FEBS Lett., 233, Matsuzawa, H., Machida, M., Kunai, K., Ito,
Y., and Ohta, T., Identification of an allosteric site residue of a
fructose 1,6-bisphosphate-dependent L-lactate dehydrogenase
of Thermus caldophilus GK24: production of a non-allosteric
form by protein engineering, 375–378, Copyright Elsevior
(1988).
Problems
1. Explain the difference between hydrogen atom, proton, and
hydride ion.
2. As for the TPP action, the importance of tautomerization of
the pyrimidine ring is mentioned. Explain tautomerization
(see Fig. 7.8c).
References
1. Fischer, J. D., Holliday, G. L., Rahman, S., Thornton, J. M., and Janet, M.
(2010). The Structures and physicochemical properties of organic
cofactors in biocatalysis. J. Mol. Biol., 403, 803–824.
2. Harden, A., and Young, W. J. (1906). The alcoholic ferment of yeast

juice. Part II. The co-ferment of yeast-juice. Proc. Roy. Soc., B., 78,
369–375.
3. Harden, A. (1914). Alcoholic fermentation. In: Monographs on
Biochemistry. (ed. Plimmer, R. H. A., and Hopkins, F. G.), Longmans,
Green and Co.
4. Fraaije, M. W., and Mattevi, A. (2000). Flavooenzyme: Diverse
catalysts with recurrent features. TIBS, 25, 126–132 (review).
5. Toney, M. D. (2005). Reaction specificity of PLP enzymes. Arch.
Biophys. Biochem., 433, 279–287 (review).
6. Scott, J.M. (1999). Folate and vitamin B12. Proc. Nutrit. Soc., 58,
441–448 (review).
7. Kluger, R., and Tittmann, K. (2008). Thiamin diphosphate catalysis:
Enzymic and nonenzymic covalent intermediates. Chem. Rev., 108,
1797–1833.
8. Adina-Zada, A., Zeczycki, T. N., and Attwood, P. V. (2012). Regulation
of the structure and activity of pyruvate carboxylase by acetyl CoA.
Arch. Biochem. Biophys., 519, 118–130 (review).
9. Davidson, V. L. (2007). Protein-derived cofactors. Expanding the
scope of post-translational modifications. Biochemistry, 46,
5283–5292 (review).
10. Janes, S. M., Mu, D., Wemmer, D., Smith, A. J., Kaur, S., Maltby, D.,
Burlingame, L., and Klinman, J. P. (1990). A new cofactor in eukaryotic
enzymes: 6-Hydroxydopa at the active site of bovine amine oxidase.
Science, 248, 981–987.
11. Means, G. E., and Feeney, R. E. (1971). Chemical Modification of
Proteins (Holden-Day, Inc. San Francisco, USA).
12. Tawfik, D. S. (1996). Side-chain selective chemical modifications of
proteins. The protein protocols handbook (ed. Waker J. M.), Humana
Press Inc., Totowa, NJ. pp. 349–368.
13. Takahashi, K. (1968). The reaction of phenylglyoxal with arginine
residues in proteins. J. Biol. Chem., 243, 6171–6179.
14. Bjerrum, P. J., Wieth, J. O., and Borders, C. L. Jr. (1983). Selective
phenylglyoxalation of functionally essential arginyl residues in the
erythrocyte anion transport protein. J. Gen. Physiol., 81, 453–484.
15. Wood, T. D., Guan, Z., Borders, C. L. Jr., Chen, L. H., Kenyon, G. L., and
Mclafferty, F. W. (1998). Creatine kinase: Essential arginine residues
at the nucleotide binding site identified by chemical modification
and high-resolution tandem mass spectrometry. Proc. Natl. Acad. Sci.
U. S. A., 95, 3362–3365.
References 139
16. Mukouyama, E. B., Hirose, T., and Suzuki, H. (1998). Chemical

modification of l-phenylalanine oxidase from Pseudomonas sp. P501
by phenylglyoxal. Identification of one essential arginyl residue.
J. Biochem., 123, 1097–1103.
17. Ida, K., Suguro, M., and Suzuki, H. (2011). High resolution X-ray
crystal structures of l-phenylalanine oxidase (deaminating and
decarboxylating) from Pseudomonas sp. P-501. Structures of the
enzyme-ligand complex and catalytic mechanism. J. Biochem., 150,
659–669.
18. Matsuzawa, H., Machida, M., Kunai, K., Ito, Y., and Ohta, T.
(1988). Identification of an allosteric site residue of a fructose
1,6-bisphosphate-dependent l-lactate dehydrogenase of Thermus
caldophilus GK24: production of a non-allosteric form by protein
engineering. FEBS Lett., 233, 375–378.
Chapter 8
Control of Enzyme Activity
Enzymes are involved in our lives in various ways and are essential
to maintain our body. Cellular components are kept in almost
constant concentrations, and pH of our body fluid and our body
temperature are also maintained at almost constant values. The
phenomena are called homeostasis. Enzymes maintain homeostasis,
since they catalyze and regulate reactions in cells. Enzyme activities
change various conditions: pH, temperature, ligand-binding, and
covalent modifications. The amount of enzyme also regulates the
reactions in cells. This chapter focuses on the control of enzymatic
activities by non-covalent ligand binding and the covalent
modification of enzyme.
8.1 Regulation by Non-Covalent Interaction

There is a metabolic pathway starting from substrate A to an end
product F (Fig. 8.1). When the end product F is overproduced, the
product F inhibits an enzyme “a.” This type of regulation is called
feedback inhibition. By inhibiting the first step of the pathway,
from A to B, a useless accumulation of metabolic intermediates of
the pathway will be prevented. A good example of the case is the
inhibition of aspartic transcarbamoylase (ATCase) by CTP (Yates
and Pardee, 1956) [1]. ATCase catalyzes the formation of N-

Haruo Suzuki
www.panstanford.com
142 Control of Enzyme Activity
carbamoyl-l-aspartate and phosphate from carbamoylphosphate

and l-Asp, the first step of pyrimidine biosynthesis. The enzyme
is inhibited by the end-product of the pathway [2]. The enzyme
like ATCase has an allosteric site in addition to the active site. The
allosteric site binds a ligand (allosteric effector) structurally different
from substrate and regulates the activity of enzyme. The word
“allo-” means “different” or “other.” The allosteric effector is generally
a substance with low molecular weight and controls the affinity
and reactivity of enzyme with substrate [3]. This type of interaction
is named heterotropic interaction. The feed forward activation in
Fig. 8.1 is a case that substrate A stimulates the activity of an
enzyme “c.” A good example is the activation of pyruvate kinase
(PK) by fructose 1,6-bisphosphate (FBP) [4]. The enzyme will be
mentioned in detail later.
Figure 8.1 Metabolic pathway. Feedback inhibition and feed forward

activation. The reactions are catalyzed by enzymes, “a”
to “e.” The end product F inhibits the enzyme “a,” and the
substrate A activates the enzyme “c.”
The allosteric enzyme contains generally more than two

binding sites of substrate which sites are identical each other.
Binding of substrate to one of the sites induces the conformational
changes of enzyme to change the affinity of the second site of
substrate binding positively or negatively. A typical case of this
type is the binding of oxygen to hemoglobin, though hemoglobin is
not enzyme. Hemoglobin is composed of four subunits, and each
subunit contains one oxygen-binding site. Binding of one molecule
of oxygen to one of the four sites accelerates the binding of oxygen
to the other subunit. This type of allosteric behavior is called
homotropic effect, since the allosteric ligand is oxygen (substrate)
itself. Figure 8.2 illustrates how the allosteric enzyme works.
Regulation by Non-Covalent Interaction 143
Figure 8.2 Schematic illustration how allosteric enzymes work. When

an allosteric effector (inhibitor) binds the allosteric site of
enzyme, the conformation of active site changes not to bind
substrate, thus inhibits the activity. In the absence of the
inhibitor, the enzyme is in the active form to form the product.
The allosteric activation is the change of the active site from
the inactive conformation to the active one by binding of the
allosteric effector (activator).
Allosteric enzymes show a sigmoidal curve when an activity

is plotted against [substrate] as shown by curves b and c in
Fig. 8.3A. Curve a is a Michaelis–Menten-type plot of activity vs.
[substrate]; non-allosteric enzymes show this type. In the case of
ATCase, a plot of the activity of enzyme vs. [Asp] shows a curve b
(homotropic interaction), but curve c in the presence of CTP [2].
The allosteric behavior observed with ATCase in the presence of
CTP is called heterotropic effect, since a ligand (CTP) is different
from substrates.
A sigmoidal curve in the plot of v vs. [S] is explained from
the following reaction:
k k

E + nS 
1
 ESn  E + nP (8.1)
k  1
This reaction means that n molecules of substrate bind

simultaneously with one molecule of enzyme. The rate of reaction
v is
V , (8.2)
v = k[ESn ]=
(K /[S]n +1)
n

where V and K are the maximum rate of reaction and the
concentration of substrate at the half maximum rate, respectively.
Equation 8.2 is called the Hill equation, named after A. V. Hill
(1910), who introduced this equation to explain the sigmoidal
curve of oxygen-binding to hemoglobin [5]. From Eq. 8.2,
v [S]n
= n (8.3)
V –v K
v [S] n
=
V – vof K n [5]. An alternative
In Eq. 8.3, K was originally used in place
expression of Eq. 8.3 is
 v 
log = n log[S]– n log K (8.4)
V – v 
(A) (B)
Figure 8.3 (A) Enzyme activity vs. [Substrate] curve observed with a
non-allosteric enzyme (a), and allosteric enzymes (b, c). In the
ATCase-catalyzed reaction, a plot of activity vs. [Asp] shows
curve b, and the same plot in the presence of CTP shows
curve c. (B) Hill plot.
The plot between log {v/(V – v)} vs. log [S] gives a linear line
(Hill plot, Fig. 8.3B). The slope of the plot gives “n,” which is called
the Hill coefficient, and a measure of cooperative binding of a ligand

to an enzyme (protein). When n = 1, the Michaelis–Menten-type
binding applies. When n > 1, one ligand is bound to the enzyme,
its affinity for the same ligand increases. When n < 1, one ligand is
bound to the enzyme, and its affinity for the same ligand decreases.
Hill coefficient is very useful to see if the enzyme is allosteric or
not. Then how can we explain the allosteric behavior of proteins
(enzymes)? The following two mechanisms are well known and
explained schematically using tetrameric enzyme (protein) in
Fig. 8.4.
Concerted model (MWC model) [3]. The model was developed
to explain the cooperative binding of oxygen to hemoglobin.
Allosteric proteins are assumed to be in a rapid equilibrium between
an active R (relaxed) and an inactive T (tense) states. The T and
R states are in a different quaternary structure (conformation).
Binding of substrate to T state leads T to R transition. Hemoglobin is
composed of four subunits, and the sigmoidal oxygen saturation
curve is well explained by this mechanism. Recent reviews are
published as the 50th anniversary of this model [6].
(a)
(b)
Figure 8.4 Two models for the binding of ligands to allosteric protein.
Tetrameric protein is modeled. S, ligand. In sequential
model, when a ligand binds to a subunit, model assumes
conformational changes of the subunit and the near-by
subunits. The conformational change of the near-by subunits
is represented by dots.
Sequential model (KNF model) [7]. Binding of a ligand to one of

the subunits induces conformational changes of the subunit, which
leads to conformational changes of the nearby contacting subunit(s).

The conformational change affects the reactivity of ligand to
the subunit. These changes are induced sequentially. The model
also explains well the oxygenation of hemoglobin. The detailed
explanations of these two models are described in [3, 6, 7].
The above models assume an oligomeric enzyme. However,
monomeric enzymes were reported to show allosteric behaviors,
which were explained kinetically [8]. Monomeric enzymes displaying
co-operativity are, for example, ribonucleotide triphosphate
reductase, glucokinase, d-chymotrypsin, and cytochrome P450.
Here, the allostery of monomeric enzymes will not be mentioned
further. Therefore, readers are referred to papers in references [8, 9].
Figure 8.5 Glycolysis. Pyruvate kinase is activated by fructose 1,6-bisphos-

phate and by phosphoenolpyruvate. Glucose is metabolized by
the initial 6 carbons and the second 3-carbon steps.
Pyruvate kinase (PK) catalyzes a transformation of phosphoenol-

pyruvate (PEP) to pyruvate, a last step in the glycolytic pathway
(Fig. 8.5):
CH3C(OPO3)COO– + ADP  CH3COCOO– + ATP (8.5)

PK is a tetramer of identical subunits, and positively controlled by
fructose 1,6-bisphosphate (FBP), and a good example of the feed
forward activation of the pathway [4, 10, 11]. Figure 8.6a shows
a sigmoidal curve in the plot of activity vs. the concentration of
phosphoenolpyruvate (PEP). Addition of FBP activates the enzyme
to show a hyperbolic curve, and Hill coefficient n decreases from
2.5 to 1.1 by the activation(Fig. 8.6b). The 3D structure of PK
supports the explanation of the allosteric nature by the concerted
model.
Figure 8.6 Allosteric regulation of Phycomyces pyruvate kinase with

FBP. (a) In the absence of fructose 1,6-bisphosphate (FBS),
the activity vs. phosphoenolpyruvate (PEP) shows a sigmoid
curve. In the presence of 100 μM FBP, the activity vs. [PEP]
shows hyperbolic curve. With increasing concentrations of
FBP, 5 μM (), 10 μM (), 25 μM (), and 100 μM (), the
plot becoming hyperbolic. Adapted from Biochim. Biophys.
Acta, vol. 874, del Vale, P., de Arriaga, D., Busto, F., and Soler,
J. A study of the allosteric kinetics of Phycomyces pyruvate
kinase as judged by the effect of l-alanine and fructose 1,6-
bisphosphte. pp. 193–204. Copyright (1986), with permission
from Elsevier. (b) The Hill plot of the data in (a) in the absence
and presence of 100 μM FBP. The PEP concentration at
log v/(V – v) = 0 gives the value of K.
Figure 8.7a shows the overall structures of the inactive T and

active R states. The difference between the T and R states are clear
from the figure. Each subunit is composed of A, B, and C domains,
which are shown in Fig. 8.7b. The word domain means a conserved
independent part of a protein structure along the amino acid

sequence. Usually domain is composed of motif(s). An average size
of domains is around 100 amino acid residues, ranging from 25 to
300 amino acid residues.
(a)
(b)
Figure 8.7 3D structure of pyruvate kinase. (a) Overall structure of T

and R states. Each subunits are in different colors. T state,
E. coli enzyme (pdb: 1pky). R state, rabbit muscle M1
(pdb: 2g50). (b) Stereo view of one subunit of E. coli pyruvate
kinase (green) (pdb: 1pky; T state) superimposed to that
of yeast enzyme (light blue) (pdb: 1a3w; R state) using
a software Chimera [12]. FBP and PG (substrate analog,
phosphoglyconate) are bound to yeast enzyme, and are shown
by spheres. A, B, and C denote the domains.
The active site is in the A domain at the interface with the B

domain, and the allosteric site (FBP-binding site) is located in the
C domain at the interface with the A domain (Fig. 8.7b). As for the
subunit structure, the overall structure of the T state is very similar
to that of the R state. However, on the transition from the T to the
R states, all 12 domains undergo simultaneous and concerted
rotations, inducing the changes of the quaternary structure and the
kinetic properties.
Regulation by Covalent Modification 149
8.2 Regulation by Covalent Modification

In cells, catalytic activities of enzymes are also controlled by covalent
modifications of proteins, such as cleavage of peptide bonds and
modification of amino acid side chains (Table 8.1).
Table 8.1 Covalent modification of proteins
Cleavage of peptide bonds Modification of amino acid residues

Cleavage of initiator Met (fMet) Phosphorylation: Ser, Thr, Tyr
Removal of signal peptide Adenylylation : Tyr
preproinsulin, pretrypsinogen ADP-riboxylation : Arg, Glu
Activation of zymogen Ubiquitination : Lys
trypsinogen, chymotrypsinogen Nitrosylation: Cys
pepsinogen, proinsulin
8.2.1 Activation of Enzymes by Cleavage of Polypeptide

Chain
Enzymes and proteins are known to be activated by cleavage of
peptide bond: activation of digestive enzymes, blood clotting, skin
formation, apoptosis, etc. As shown in Table 8.1, various enzymes
are expressed as proenzyme (zymogen) and activated by the
proteolytic cleavage of prosequence. Here, we focus on the activation
of trypsinogen.
Bovine trypsinogen is expressed as a proform of a single
polypeptide chain of 246 amino acid residues length in pancreatic
acinar cells (Fig. 8.8). The initiator Met must be removed during
the elongation of the polypeptide chain on polysomes, and
the signal sequence from residues 2 to 17 is removed to form
trypsinogen, which is stored inside granules in the cells. The
contents containing trypsinogen in the granules are secreted into
a duct with high concentrations of HCO–3 (pH 8). The duct joins
with a common bile duct, and the contents (pancreatic juice) flow
together, releasing into the duodenum. Trypsinogen secreted is
activated with enteropeptidase, which is produced by cells of the
duodedum and specifically cleaves the peptide bond between Lys23
and Ile24 of trypsinogen. The enzyme formed is named b-trypsin,
which autocatalytically cleaves at other positions. One cleaved

at Lys148-Ser149 is named a-trypsin. These two forms are major
trypsin. Trypsins cleaved at different positions are known [13].
Other pancreatic digestive enzymes, such as chymotrypsinogen,
proelastase, and procarboxypeptidase, are similarly activated.
Figure 8.8 A schematic representation of activation of bovine pancreatic

trypsinogen. Arrow heads on the top line indicate the
position of cleavage. Amino acid sequence is from BC134797
of DDBJ. Numbering of amino acid residues shown above is
applied for trypsinogen and trypsin in the text.
A question arises: Why is trypsinogen inactive? [14]. Let us

consider the question on the basis of the structures of trypsinogen
and b-trypsin. Figure 8.9 shows 3D structures of trypsin and its
complex with trypsin inhibitor. The carboxyl group of Asp194 in
the bottom of the specificity pocket of trypsin electrostatically
interacts with the side chain amino group of Lys 15 of the inhibitor.
Moreover, the active site Triad is covered by the inhibitor. Thus, the
substrate of trypsin must enter from the right side of trypsin in
its structure shown in Fig. 8.9a. If the prosequence of trypsinogen
covers the active site and entrance of substrate like trypsin
inhibitor, we easily understand the inactivity of trypsinogen.
However, this is not the case. Figure 8.10 shows 3D structures of
(a) (b)
Figure 8.9 Cartoon representation of trypsin (a) and trypsin-trypsin

inhibitor complex (b). Trypsin (pdb 3tgi) is shown magenta,
and bovine pancreatic trypsin inhibitor is blue. Pdb code
for the complex is 1qcp. Active site triad, Ser200, His63,
and Asp107 are shown yellow spheres. Asp194 of trypsin
is shown cyan spheres. Lys15 of the inhibitor is shown blue
spheres. The active site is capped with the inhibitor.
Figure 8.10 A schematic representation of activation of bovine

trypsinogen. Pdb codes of trypsinogen and trypsin are 1tgt
and 3ptb, respectively. Ile24 (I24) is on the surface of the
molecule of trypsinogen, but inside of trypsin. The N-terminal
VD4K sequence of trypsinogen is not given in the pdb file,
suggesting that these residues are movable on the outside of
the molecule.
trypsinogen and trypsin. The N-terminal amino acid residues of

trypsinogen are far apart from the active site and do not cover the
active site. Thus, the substrate of trypsin seems to enter into the
active site of trypsinogen easily. Then, the previous question arises
again: why trypsinogen is not active. Though the structures of N-
terminal region of trypsinogen and trypsin are different each other,
their structures at the active site and the entrance of substrate are
quite similar each other (Fig. 8.10). However, the close-up view of
the active site and of N-terminal region of trypsinogen is different
from that of trypsin (Fig. 8.11). The amino N atom of I24 of trypsin is
hydrogen-bonded to the side chain carboxy O atom of D199, which
is linked to Ser200. On the other hand, Ile24 is far apart from the
Asp199 residue in trypsinogen. As for the distance between the
carboxy O atom of Asp194 and the imidazole N atom of His63 or
the main chain N atom of Gly198 in these proteins, the distance
(8.2 Å) between the carboxy O atom of Asp194 and the N atom of
Gly198 in trypsinogen is quite different from that in trypsin (10.9 Å).
This leads to the decrease in the size of the specificity pocket of
trypsinogen (Fig. 8.12). In addition, the distance between the N atom
of Gly198 and that of Ser200 is shorter in the trypsin (4.0 Å), but
longer in trypsinogen (4.8 Å). These residues are composed of the
oxyanion hole (see Box 8.1). Thus, trypsin is catalytically active, but
trypsinogen is not.
Figure 8.11 A close-up view of active sites of trypsinogen and trypsin.

Residue numbers follow the numbering of protrypsinogen in
Figure 8.8. Numbers near dashed line represent the distance
(Å) between atoms related. Benz represents benzamidine. In
the trypsin-benzamidine complex, amidine group interacts
with the carboxy group of Asp194, and the main chain nitrogen
of the N-terminal Ile (I24) is hydrogen-bonded with the
carboxy oxygen of Asp 199. Notice that the conformations
of Asp199 and Gly198 in trypsin are different from those in
trypsinogen.
Box 8.1
Catalytic Triad and Oxyanion Hole
Catalytic mechanism of serine protease is the same as that described
in Eq. 4.39 (Chapter 4). Formation of ES complex, which leads to a
tetrahedral reaction intermediate, and breakage of the peptide
bond to form a product and an acylated enzyme intermediate. The
acyl-enzyme intermediate is attacked by water to form another
tetrahedral intermediate, which is transformed to another product
and the original enzyme. During these reactions, the catalytic
triad is formed with Asp, His, and Ser residues, and oxyanion hole
formed with two main chain imino groups of S200 and G198
(dashed circles in the figure). Hydrogen bonding between D107
and H63 makes H63 imidazole more basic to abstract proton from
the hydroxymethyl group of S200. Thus, the negatively charged
oxygen atom of S200 attacks nucleophilically the carbonyl carbon
of peptide bond, leading to the formation of a tetrahedral intermediate
containing oxyanion. The oxyanion is stabilized by an oxyanion hole in
the enzyme-transition state (ET).
Figure 8.12 Cross-sectional view of trypsinogen and trypsin. Figures

are from the surface view of Figure 8.11. The dashed circles
show the specificity pocket, indicating the larger size of the
pocket in the trypsin-benzamidine complex.
8.2.2 Regulation by the Side Chain Phosphorylation

We learned in Chapter 7 that the chemical modification of
functional groups of enzymes changes its activity and is a good
method to study on the active site structure. Living cells regulate
their physiological states by modifying the side chain of amino
acid residues of enzyme. Table 8.1 shows some of examples of
modification. Different from the modifications in protein chemistry,
modifications in living cells are mostly reversible. We will focus on
the phosphorylation of enzymes, since it is widely known for the
regulation of metabolism: glycolysis, gluconeogenesis, glycogen
synthesis, etc. Phosphorylation of protein is catalyzed by a protein
kinase [15]. Protein kinases account for ~2% of most genomes and
include over 500 separate genes in human genome [15]. Hydroxyl
groups of specific serine, threonine, and tyrosine residues of target
enzymes are phosphorylated by protein kinase, which are grouped
into two classes, one phosphorylates specific serine or threonine
residues, and the other the specific tyrosine residue. A phosphoryl
group donor is ATP. Phosphorylation leads to the inhibition or
activation of target enzymes. The inhibition or activation depends
on the target enzyme to be phosphorylated. The phosphorylated
enzymes are recovered to the unphosphorylated form by
phosphatase (Fig. 8.13).
Figure 8.13 Phosphorylation/dephosphorylation of enzyme proteins.

Phosphorylation is catalyzed by protein kinase, and dephos-
phorylation by protein phosphatase. ATP is a phosphoryl
group donor. Protein phosphatase catalyzes the hydrolysis of
phosphoryl ester of enzyme.
The local structure surrounding the residue phosphorylated

by a particular protein kinase has a common amino acid sequence:
consensus sequence [16]. For example, protein kinase A (PKA)
phosphorylates serine or threonine residues, and the sequence
surrounding the residue is Arg-Arg-X-Ser (or Thr)-Z, where

phosphorylating residues are written bold, and X and Z are any
and hydrophobic residues, respectively. Phosphorylation of serine
residues is usually preferred over threonine residues with most
protein kinases.
8.2.2.1 cAMP-dependent protein kinase, protein kinase

A (PKA) and glycogen metabolism
Of protein kinases, PKA was first characterized in 1968 and is
involved in various processes in living cells: glycogen breakdown in
liver and muscle, ion channels, and gene expression [15]. Within the
large family of protein kinases, PKA is one of the best understood
members and thus is treated as a prototype of the entire family.
Therefore, PKA is considered here.
Figure 8.14 Model of the cAMP formation by glucagon. R and AC represent

glucagon receptor and adenylate cyclase, respectively.
G protein is heterotrimer composed of a, b and g subunits.
As for glycogen breakage, how PKA is involved. When the

glucose level decreases in the bloodstream, glucagon is secreted into
the bloodstream from pancreas. Glucagon reaches to the cells, and
binds with the glucagon receptor at the outer surface of the cells
(Fig. 8.14). The binding induces the conformational change of the
receptor, which in turn activates G protein at the inner surface of
the plasma membrane. G protein, guanine nucleotide-binding protein,
is a heterotrimeric protein (Gabg) composed of three subunits,
a, b, and g. The a subunit binds with GDP to form Gabg-GDP. The
activated G protein exchanges the bound GDP of the a subunit
with GTP. The reaction is catalyzed by guanine nucleotide exchange
factors (GEFs) [17]. The GTP-bound a subunit (Ga·GTP) is dissociated
from the bg complex (Gbg). The Ga · GTP complex activates an

adenylate cyclase, which catalyzes the following reaction:
(8.6)
The a subunit of G protein (Ga) has a GTPase activity, thus

forming Ga · GDP, which reassociates with Gbg to reform Gabg-GDP. The
GTPase activity of Ga in vitro is low to explain this cycle of G protein
in vivo. The paradox is resolved by the fact that the cells contain
GTPase-activating proteins (GAPs), which accelerate the GTPase
activity of Ga [17].
cAMP activates PKA, which phosphorylates phosphorylase
kinase. The kinase in turn phosporylates phosphorylase b (low
activity) to phosphorylase a (high activity). Phosphorylase a cleaves
the terminal glucose unit of glycogen as glucose-1-phosphate,
which is dephosphorylated to glucose in a cell. Thus, the cell can
supply glucose. In this case, the target of PKA is phosphorylase
kinase. The physiological responses depend on which enzymes are
phosphorylated. Now onward, we focus on PKA itself.
8.2.2.2 Regulatory subunit of PKA

cAMP-dependent protein kinase, protein kinase A (PKA) is a
heterotetrameric protein composed of C2R2, where C and R represent
catalytic and regulatory subunits, respectively [15]. There are three
isoforms, Ca, Cb, and Cg, in the C subunit, and four isoforms, RIa,
RIb, RIIa, and RIIb are in the R subunit (Fig. 8.15). A C2R2
complex is inactive, but binding of cAMP to each of the R subunit
leads to the dissociation of the active catalytic subunit from the
complex,
C2R2 + 4 cAMP  2C + 2R(cAMP)2 (8.7)
The binding of cAMP to the holoenzyme (C2R2 complex) is

cooperative, and the Hill coefficient and the dissociation constant
are around 1.6 and 50 nM, respectively. The R subunit contains two
domains, A and B (Figs. 8.15 and 8.16). cAMP binds B domain first,
then A domain. This is because the binding-site of the A domain is
covered by the catalytic subunit (Fig. 8.16, upper part). Figure 8.16
shows the structural change of the R subunit by binding with
cAMP. The large conformational changes of the CRIa complex are
apparent by binding with cAMP. The notable changes are observed
in the helical linkage between A and B domains, and the distance
between Arg366 and Glu261. As for the former, the linkage is
almost linear in the CR complex, but bending in the R-(cAMP)2
complex. As for the latter, the side chains of these residues are
forming the ionic bond in the CR complex, but far apart in the
cAMP-bound R subunit. Moreover, the ionic bonds tether two
adenine-capping residues, Y371 and W260, in the apo-R subunit,
but by binding with cAMP the ionic bonds are broken, leading
these residues to cap the adenine ring of cAMP.
Figure 8.15 Structural arrangements of PKA subunits. Amino acid

sequences of human PKA (P10644, P31321, P12861, P31323)
from Uniprot are arranged (18). The catalytic subunit isoforms,
a, b, and g are 350 amino acid residues long, and 78% identical
in their amino acid sequences. The phosphorylation sites,
T197 and S338 are also shown in the a subunit of catalytic
subunit (Ca). The helical region of the N-tail was from the
X-ray structure (pdb: 1j3h). In the regulatory subunit
isoforms, RIa, RIb, RIIa, and RIIb, the a isoforms are shown.
The RI and RII isoforms are 77% and 64% identical in their
amino acid sequences, respectively. Pseudo-substrate motifs
of RIa and RIIa in the linker are shown. The D/D domains,
the helical linkers (HL) are derived from the NMR solution
structures (pdb: 2ezw, 2drm), and the crystal structures
(pdb: 2qcs, 2qvs), respectively.
Figure 8.16 Mechanism of the PKA activation. The CaRIa dimeric complex
(pdb code: 2qcs) receives two molecules of cAMP, and
dissociated into the Ca and RIa · cAMP2 (pdb code: 1rgs).
Helical linker (HL) is linking two domains, and colored orange,
A domain, magenta, and B domain, pale blue, cAMP, green.
Pseudo-substrate motif (PSM) is colored gray cube.
Each R subunit contains an N-terminal dimerization/docking

(D/D) domain, a flexible linker acting as an inhibitor site, and two
tandem cyclic nucleotide-binding domains A, B (Fig. 8.15). The D/D
domain serves for dimerization of two R subunits, and for docking
with PKA anchoring proteins (AKAPs), which have a role of the
subcellular localization of PKA [19]. Probably R subunit isoforms
may determine where they work in the cell. The X ray
crystallography of PKA holoenzymes did not show the D/D domain,
indicating that the domain is movable. The solution structure
determined by the NMR method contains a helix-loop-helix motif,
which interacts with that of the other R subunit. The N-terminal
side helices of the domains are interacting with the helical region
of AKAP by hydrophobic interaction (Fig. 8.17).
Figure 8.17 The hydrophobic interaction of the D/D domain of R subunit

with the peptide derived from AKAP (pdb code: 2drn). The
figures are related approximately 90˚ rotation about a vertical
axis. The hydrophobic residues of the helix in the AKAP
peptide and the N-terminal side-helical regions are shown in
black.
The pseudo-phosphorylation site in the flexible linker of

the R subunit contains a sequence quite similar to that of the
phosphorylation site of the PKA substrate (the pseudo-substrate
motif: RRXSZ) (Fig. 8.15). The amino acid sequences around the
pseudo-phosphorylation site of human R subunits are
RIa GRRRRGAISAE
RIb ARRRRGGVSAE
RIIa RFVRRVSVCAE
RIIb RFTRRASVCAE
The amino acid residue of the site in RII isoforms is Ser, but Ala
or Gly in RI isoforms. In the RC complex, the pseudo-phosphorylation
site occupies the active site of the C subunit like the trypsin-trypsin
inhibitor complex (Figs. 8.9 and 8.16), leading to the strong binding
of a C subunit with an R subunit.
8.2.2.3 Catalytic subunit and overall reaction mechanism of

catalysis
The catalytic subunit (C subunit) is bilobal protein, the N-terminal
N-lobe and the C-terminal C-lobe (Fig. 8.18a). The active site is
located at the cleft between two lobes. The large C-lobe is helical and
has the role of phosphoryl transfer, and the small N-lobe is b rich
and has ATP-binding site.
(a)
(b)
Figure 8.18 Crystal structure of the catalytic subunit of PKA. (a) Apoform
(open form. pdb code: 1j3h). (b) Inhibitor peptide (IP)-bound
form (closed form. pdb code:3fjq). In the C-tail, the residues
from 319 to 328 is missing in the open form (a), but clearly
seen in the closed form (b). The inhibitory peptide (IP) is
shown in orange cartoon, and the RRNAI sequence in red sticks.
The phosphorylated T197 and S338, F318, and D329 are blue
spheres, and ATP green sticks. F327 and Y330, red sticks; N-
tail, green; N-lobe, magenta; C-lobe, cyan; C-tail, red.
The C-tail wraps around both lobes, and the residues from
319 to 328 must be movable, since these residues are not observed
by the X ray crystallography in the apo C subunit (Fig. 8.18a). On
the other hand, by binding with ligands, ATP and inhibitory peptide
(IP in Fig. 8.18b), these residues become structurally visible, and
F327 and Y330 in the region trap ATP cofactor. The N-tail contains
helical structure (aA), which interacts with the PKA interacting
protein 1(AKIP1) in the nucleus. The C subunit contains two
phosphorylation sites, S338 and T197, and the phosphorylation of

these residues is required for the full activity of the C subunit.
The pre-steady state analyses of the C subunit-catalyzed
phosphorylation of a short substrate (S: Kemptide, Fig. 8.19) showed
the rapid formation of the phosphorylated Kemptide (P) (Fig. 8.20)
[20]. After this short period (approximately 10 ms), a slow linear
increase of the product follows. Extrapolation of the slow phase to
the time zero gives the intercept (p) on the ordinate, which increases
with the increase of the C subunit concentration (see Box 8.2). The
ratio of the p value at infinite concentration of substrate to the
subunit concentration gives the constant value of 1.
Figure 8.19 Amino acid sequences of substrates and its analog for PKA. The
sequences of SP20 and IP (inhibitor peptide) are numbered
according to the sequence number of the inhibitor for PKA.
Figure 8.20 Initial burst of the phosphorylated Kemptide in the PKA

reaction. The concentration shown near each line is that of the
C subunit used. The conditions, 200 μM [g-P32] ATP, 10 mM free
Mg2+, 100 μM Kemptide, in 50 mM MOPS (pH 7.0). Adapted
with permission from Grant, B. D., and Joseph A., Adams, J. A.
Pre-steady-state kinetic analysis of cAMP-dependent protein
kinase using rapid quench flow techniques. Biochemistry, 35,
2022–2029. Copyright (1996) American Chemical Society.
Box 8.2
Initial Burst and Active Site Titration
In the enzymatic reaction to form two products, when one product
(P1) is rapidly formed, followed by a slow formation of the second
product (P2), the rapid formation of P1 is called “initial burst.” The
phenomena had been observed for the protease-catalyzed hydrolysis
of esters.
Observation of the phenomena is the good evidence for the

following reaction scheme:
k +1 k k

E + S   ES 
+2 +3
 ES   E + P2
k –1 (B8.1)
+ P1

From the scheme, we can introduce the following equation of the
time-dependent formation of P1.
The concentration of P1, [P1] = p1 is
p1 = v0t + p(1 – e – t ) (B8.2)
where
k+2k+3e0 s /( k+2 + k+3 ) (B8.3)

v0 =
s + Km
2 2
 k+2  s 
p =   e0 (B8.4)
 k+2 + k+3  K m + s 
k+2 s
 = k+3 + (B8.5)
(K + s )
where e0, s, K, and Km represent the initial concentration of enzyme,
the concentration of substrate, the dissociation constant of the ES
complex, and Michaelis constant, respectively, and s » e0. The intercept
__
on the ordinate of the plot of 1/√p vs. 1/s gives e0 when k+2 » k+3. The
value obtained is the active site concentration of enzyme, leading to
the good method for the determination of the concentration of active
enzyme.
In the case of myosin ATPase-catalyzed “rapid” liberation of organic

phosphate, phenomena are similar to those observed with protease,
though the reactions follow apparently two steps mechanism under
undenatured conditions. From the present knowledge of this reaction,
the phenomena were observed because reactions were stopped by the
addition of denaturant (trichloroacetic acid), just like the quenched
flow method performed with PKA described in the text. The myosin
ATPase reaction follows:
k k+2 k+3
M + ADP + P i, (B8.6)
+1

M + ATP   M ATP  M ADPP i 
k –1
where M represents myosin ATPase. The M·ADP·Pi complex is formed

rapidly, but the product Pi is still bound to M. Therefore, the overall
formation of Pi is limited by the slow next step (k+3). When the
reaction was stopped by the addition of trichloroacetic acid, then
the product Pi was released from M. Thus, rapid formation of Pi was
observed, and the phenomena are also called initial burst.
The kinetic behavior like this has been known as “Initial Burst”
in protease-catalyzed ester hydrolysis and in myosin ATPase
reaction [21, 22]. Thus, the phosphoryl transfer reaction of
PKA can be explained similarly. The C-subunit-ATP complex
forms the Michaelis complex with Kemptide (S), which rapidly
changes to the C-subunit-ADP-P complex. The C subunit-ADP-P
complex releases ADP and P to reform the C-subunit. According
to Grant and Adams, the rate of the phosphoryl transfer is 500 s–1,
and that of the release of products is 21 s–1,
–1 –1
500 s 21s

C ATP + S  C ATPS 
  C ADPP   C + ADP + P (8.5)
In the scheme, C represents the C-subunit, S the substrate Kemptide,

and P the phosphorylated Kemptide. The scheme explains the
overall reaction of the C-subunit-catalyzed phosphorylation of
Kemptide.
However, the detailed mechanism of the phosphoryl transfer
is under debate. Magnesium ion neutralizes the negative charge of
ATP in the ATP-Mg complex. However, studies on the C-subunit
in complex with ATP, peptide substrate, and/or Mg showed that
Mg ion has the specific role in the catalytic reaction [23]. That is, the
active site has two binding sites of Mg ion, Mg1 and Mg2, and that
the binding to the Mg1 site is weak. From the structural and kinetic
studies, a catalytic cycle of PKA is proposed (Fig. 8.21) [23]. Release
of Mg ion from the Mg1 site is prerequisite for the release of ADP-
Mg. A catalytic cycle similar to that presented Fig. 8.21 has been
proposed for cyclin-dependent kinase [24].
Figure 8.21 Catalytic cycle of protein kinase A. Reproduced with

permission from Bastidas, A. C., Deal, M. S., Steichen, J. M.,
Guo, Y., Wu, J. and Taylor, S. Phosphoryl transfer by protein
kinase a is captured in a crystal lattice. J. Am. Chem. Soc. 135,
4788–4798. Copyright (2013) American Chemical Society.
8.2.2.4 Phosphoryl transfer reactions at the active site of the

C subunit
Phosphorus locates just above nitrogen in the periodic table and is
on the third row of the table. Thus, it forms stable 3, 4, and 5 co-
ordinated compounds. As for the phosphoryl transfer reaction from
R2-OPO–2
3 to R1OH,
3  R1-OPO3 + R2OH

R1OH + R2-OPO2– (8.6)
2–
Two mechanisms of the phosphoryl transfer reaction seem to be

widely accepted (Fig. 8.22). One is the dissociative mechanism, a
SN1-like reaction in the carbon chemistry. Here “S” means
“substitution,” and “SN” stands for the nucleophilic substitution and
“1” represents the fact that the rate follows the first-order rate law
(Chapter 5). The reaction first eliminates the R2O– group to form
the unstable metaphosphate intermediate. The second one is the
associative mechanism, a SN2-like reaction, that is, the rate follows
the second-order rate law. The R1O– group attacks the P atom of
the R2-phosphate to form the unstable pentavalent phosphate
intermediate. Uncatalyzed hydrolysis of ATP in solution occurs
via dissociative, metaphosphate-like transition state [25].
Figure 8.22 Transphosphorylation mechanisms. B: nucleophile. R1OH,

accepter; R2OPO2–
3
, donor.
Computational studies on the mechanism of the C-subunit-

catalyzed phosphoryl transfer reaction support that the reaction
proceeds through the dissociative-like transition state shown
below [26–28].
There are many works on the C-subunit-catalyzed phosphoryl

transfer reaction [15]. Here, the works by Dr. Taylor’ group will be
introduced [23]. Usually, adenylyl imidodiphosphate (AMP-PNP)

and a, b-methylene adenosine diphosphate (AMP-PCP) have been
used as a non-hydrolysable ATP analog. Unexpectedly, AMP-PNP is
hydrolyzed during the crystallization of the C-subunit.
After three months of crystallization, Bastidas et al. [23] found two

crystal structures. One structure displays two states of protein:
55% of the protein contains AMP-PNP and a substrate peptide
SP20, and 45% contains AMP-PN and a phosphorylated SP20.
These results are informative on the molecular mechanism of the
phosphoryl transfer, showing structures of the midpoint and the
endpoint of the reaction.
(a)
(b)
Figure 8.23 Stereo views of the active site of the C-subunit of PKA. (a) The
crystal structure of the C-subunit in complex with AMP-PNP
(thin stick), AMP-PN (thick stick), Mg, and SP20 (pdb: 4hpu).
(b) The transition state mimic of the C-subunit in complex with
ADP, AlF3, and SP20 (pdb: 1l3r).
References 167
In the C-subunit-catalyzed phosphoryl transfer reaction, the

conserved Asp166 has been assumed to be a nucleophile. If this
is the case, the carboxy O atom must be faced to the side chain
of Ser21 of SP20. However, the side chain of Ser21 is away from
Asp166 before and after the phosphoryl transfer as shown in Fig.
8.23a. That is, the distance from the side chain O atom of Ser21
to the carboxy O atom of Asp166 is 4.7–5.9 Å. This contradiction
may be explainable from the structural and computational studies
[23]. Figure 8.23b shows a stereo view of a transition state mimic.
The distance from the side chain O atom of Ser21 to the carboxy
O atom of Asp166 is around 2.5–3.5 Å, suggesting that in the
transition state Asp166 serves as a nucleophile to receive proton
from Ser21 of SP20. This is supported by the computational studies
[26–28].
Problems
1. Demonstrate that K is the substrate concentration at the
half-maximum rate in the Hill equation.
2. Figure 8.21 shows the catalytic cycle of C-subunit-catalyzed
protein kinase reaction. Two magnesium ions bind and
release from the enzyme. Based on the mechanism, draw the
scheme by the Cleland-type expression, including magnesium
ions, though magnesium ions are neither substrate nor
product.
3. From the scheme in Eq. B8.1 in Box 8.2, derive Eq. B8.2 by
assuming rapid equilibrium (see Chapter 2).
__
4. In the Box 8.2, how does a plot between 1/√p and 1/s lead
to give the active concentration of enzyme?
References
1. Yates, R. A., and Pardee A. B. (1956). Control of pyrimidine biosynthesis

in Escherichia coli. J. Biol. Chem., 221, 757–770.
2. Lipscomb, W. N., and Kantrowitz, E. R. (2012). Structure and
mechanisms of Escherichia coli aspartate transcarbamoylase. Acc.
Chem. Res., 20, 444–453 (review).
3. Monod, J., Wyman, J., and Changeux J. P. (1965). On the nature of
allosteric transitions: A plausible model. J. Mol. Biol., 12, 88–118.
4. del Vale, P., de Arriaga, D., Busto, F., and Soler, J. (1986). A study of the
allosteric kinetics of Phycomyces pyruvate kinase as judged by the
effect of l-alanine and fructose 1,6-bisphosphate. Biochim. Biophys.
Acta., 874, 193–204.
5. Hill, A. V. (1910). The possible effects of the aggregation of molecules
of hemoglobin on its dissociation curves. J. Physiol., 40, 4–7.
6. Allosteric interactions and biological regulation (part I and II) (2013).
J. Mol. Biol., 425, 1391–1592, and pp. 2277–2392, edited by Kalodimos,
C., and Edelstein, S. (reviews).
7. Koshland D. E. Jr., Némethy, G., and Filmer, D. (1966). Comparison
of experimental binding data and theoretical models in proteins
containing subunits. Biochemistry, 5, 365–385.
8. Cornish-Bowden, A., and Cárdenas, M. L. (1987). Co-operativity in
monomeric enzymes. J. Theor. Biol., 124, 1–23 (review).
9. Denisov, I. G. and Sligar, S. G. (2012). A novel type of allosteric
regulation: Functional cooperativity in monomeric proteins. Arch.
Biochem. Biophys., 519, 91–102.
10. Jurica, M. S., Mesecar, A., Heath, P. J., Shi, W., Nowak, T., and Stoddard, B.
L. (1998). The allosteric regulation of pyruvate kinase by fructose-1,6-
bisphosphate. Structure, 6, 195–210.
11. Mattevi, A., Valentini, G., Rizzi, M., Speranza, M. L., Bolognesi, M.,
and Alessandro Coda, A. (1995). Crystal structure of Escherichia coli
pyruvate kinase type I: Molecular basis of the allosteric transition.
Structure, 3, 729–741.
12. Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt,
D. M., Meng, E. C., Ferrin, T. E. (2004). UCSF Chimera: A visualization
system for exploratory research and analysis. J. Comput. Chem., 25,
1605–1612.
13. Walsh, K. A., and Wilcox, P. E. (1970). Serine proteases. In Methods in
Enzymology (ed. Colowick, S. P., and Kaplan, N. O.) 19, 31–63 (review).
14. Stroud, R. M., Kossiakoff, A. A., and Chambers, J. L. (1977). Mechanism of
zymogen activation. Ann. Rev. Biophys. Bioeng., 6, 177–193 (review).
15. Taylor, S. S., Ilouz, R., Zhang, P., and Kornev, A. P. (2012). Assembly of
allosteric macromolecular switches: Lessons from PKA. Nat. Rev. Mol.
Cell Biol., 13, 640–658 (review).
16. Ubersax, J. A., and Ferrell, J. E. (2007). Mechanism of specificity in
protein phosphorylation. Nat. Rev. Mol. Cell Biol., 8, 530–541.
17. De Vries, L., Zheng, B., Fischer, T. Elenko, E., and Farquhar M. G. (2000).
The regulator of G protein signaling family. Annu. Rev. Pharmacol.
Toxicol., 40, 235–271 (review).
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18. The UniProt Consortium (2013). Update on activities at the Universal

Protein Resource (UniProt) in 2013. Nucleic Acids Res., 41, D43–D47.
19. Pidoux, G., and Taskén, K. (2010). Specificity and spatial dynamics of
protein kinase A signaling organized by A-kinase-anchoring proteins.
J. Mol. Endocrinol., 44, 271–284 (review).
20. Grant, B. D., and Joseph A., Adams, J. A. (1996). Pre-steady-state kinetic
analysis of cAMP-dependent protein kinase using rapid quench flow
techniques. Biochemistry, 35, 2022–2029.
21. Hartley, B. S., and Kilby, B. A. (1954). The reaction p-nitrophenyl
esters with chymotrypsin and insulin. Biochem. J., 56,
288–297.
22. Kanazawa, T., and Tonomura, Y. (1965). The pre-steady state of the
myosin-adenosine triphosphate system. I. Initial rapid liberation of
inorganic phosphate. J. Biochem., 57, 604–615.
23. Bastidas, A. C., Deal, M. S., Steichen, J. M., Guo, Y., Wu, J. and Taylor,
S. (2013). Phosphoryl transfer by protein kinase a is captured in a
crystal lattice. J. Am. Chem. Soc., 135, 4788–4798.
24. Jacobsen, D. M., Bao, Z.-Q., O’Brien, P., Brooks, C., Brooks, C. L.,
and Young, M. (2012). Price to be paid for two-metal catalysis:
Magnesium ions that accelerate chemistry unavoidably limit product
release from a protein kinase. J. Am. Chem. Soc., 134, 15357–15370.
25. Admiral, S. J., and Herschlag, D. (1995). Mapping the transition state
for ATP hydrolysis: Implication for enzymatic catalysis. Chem. Biol., 2,
729–739.
26. Cheng, Y., Zhang, Y., and McCammon, A. J. (2005). How does the cAMP-
dependent protein kinase catalyze the phosphorylation reaction: An
ab initio QM/MM study. J. Am. Chem. Soc., 127, 1553–1562.
27. Valiev, M., Yang, J., Adams, J. A., Taylor, S. S. and Weare, J. H. (2007).
Phosphorylation reaction in cAPK protein kinase-Free energy quantum
mechanical/molecular mechanics simulations J. Phys. Chem., 111,
13455–13464.
28. Montenegro, M., Garcia-Viloca, M., Lluch, J. M., and Àngels González-
Lafont, A. (2011). A QM/MM study of the phosphoryl transfer to the
Kemptide substrate catalyzed by protein kinase A. The effect of the
phosphorylation state of the protein on the mechanism. Phys. Chem.
Chem. Phys., 13, 530–539.
Chapter 9
Preparation of Enzyme
We learned about kinetic and structural aspects of enzymes

and how they regulate the metabolic pathways in the cell. When it
comes that we start to work on an enzyme, we must have the enzyme.
The enzyme of interest may be available commercially, or from
other researchers. Even in these cases, you must test the purity of
the enzyme. This chapter summarizes the methods for the
purification of enzymes and the purity analysis. The methods
described are rather qualitative and not in detail; therefore, for
further information, see [1, 2].
9.1 Extraction of Enzyme

For the purification of the enzyme, we must take care of the
following points in the purification procedures. (1) Temperature
should be kept as low as possible, around 5°C, since most proteins
are denatured at high temperature. (2) Buffers used for the
purification are better to be around pH 7, since most proteins are
stable under neutral conditions. As an example, steps of preparation
of extracts from liver are described.
It is better to get the fresh liver, such as from a slaughterhouse.
The liver should be cooled as soon as possible. Then, prepare
the extract in a cold room (4–5°C). Cut the liver into small pieces
(smaller than 1 cm length in width) and homogenize these in a

Haruo Suzuki
www.panstanford.com
172 Preparation of Enzyme
buffer using an apparatus such as blender, Potter–Elvehjem homo-

genizer, sonicator, and French press.
A blender is familiar in kitchen to prepare juices of vegetables
and fruits. It is useful for relatively large-scale homogenization.
The Potter-Elvehjem homogenizer is one of a mortar-pestle type
homogenizer and is a widely used apparatus. It has been named
after van R. Potter and C. A. Elvehjem. The mortar is made of a
cylindrical glass, and the pestle is made of Teflon. The pestle rotates
by an electric motor, while small tissues in buffer go through narrow
interface between the mortar and the pestle; thus, the tissues are
disrupted.
The sonicator uses the energy of the ultrasonic sound to
disrupt tissues (cells), and is used for a small-scale to large-scale
homogenization. However, it is not recommended to use tough
tissues. During sonication, it is important to cool the homogenization
mixture, since heat is generated during sonication.
French press is a method to lyse cells using a high pressure,
named after C. S. French. It is useful for the small-scale homo-
genization. The cell suspensions to be disrupted are passed through
narrow valve under high pressure, and exposed to atmosphere.
This causes a large pressure drop, inducing the disruption of cells.
During homogenization, samples should be cooled as
described above. In addition, buffers used are better to include
chemicals to prevent the action of proteases. Some proteases
require metal ion; therefore, EDTA (ethylenediamine tetraacetic
acid) is better to be added to chelate metal ions. Moreover, a serine
protease inhibitor, phenylmethane sulfonylfluoride (PMSF), is
also better to be added.
9.2 Purification of Enzyme

9.2.1 Method to Use the Solubility of Proteins
The method is used in the initial stage of purification and to
concentrate the proteins.
9.2.1.1 Salting-out
Most proteins are soluble in water under low ionic strength,
but insoluble at high ionic strength. This precipitation is called
salting out. Its principle is briefly as follows. Proteins in water
Purification of Enzyme 173
are surrounded by water molecules, which interact with amino

acid residues of protein. When the salt is added to the protein
solution, some of the water molecules are used to dissolve the
salts, resulting in the decrease in the water molecules that
are interacting with the amino acid residues on the surface of
proteins. These lead to the increase of protein-protein interaction
to precipitate.
Table 9.1 Grams of ammonium sulfate necessary to take 1 liter solution

from one “percent saturation” to another
Final concentration of ammonium sulfate, % saturation

10 20 25 30 33 35 40 45 50 55 60 65 70 75 80 90 100
Grams solid ammonium sulfate to be added to 1 liter of solution
0 56 114 144 176 196 209 243 277 313 351 390 430 472 516 561 662 767
10 57 86 118 137 150 183 216 251 288 326 365 406 449 494 592 694
Initial concentration of ammonium sulfate,
20 29 59 78 91 123 155 189 225 262 300 340 382 424 520 619
25 30 49 61 93 125 158 193 230 267 307 348 390 485 583
30 19 30 62 94 127 162 198 235 273 314 356 449 546
33 12 43 74 107 142 177 214 252 292 333 426 522
35 31 63 94 129 164 200 238 278 319 411 506
% saturation
40 31 63 97 132 168 205 245 285 375 469

45 32 65 99 134 171 210 250 339 431
50 33 66 101 137 176 214 302 392
55 33 67 103 141 179 264 353
60 34 69 105 143 227 314
65 34 70 107 190 275
70 35 72 153 237
75 36 115 198
80 77 157
90 79
Source: Reproduced with permission from Methods Enzymol., 1, Green, A. A., and
Hughes, W. L. Protein fractionation on the basis of solubility in aqueous solutions of
salts and organic solvents, 67–90, Copyright Elsevier (1955).
Ammonium sulfate is commonly used for the salting out of

proteins, since the solubility is almost independent of temperature
(70–80 g/100 g water at 0 to 40°C), indicating high solubility at
low temperature. It is better to add solid ammonium sulfate to
the protein solution for keeping the increase of the volume of
the protein solution as small as possible. The ammonium sulfate
solution is acidic, so care must be taken to keep the solution
neutral by the addition of ammonia solution. It may be better to
start by the addition of ammonium sulfate from 10% saturation.

When precipitates appeared, keep the mixture for 10 min or
more, and collect the precipitates by centrifugation.
To the supernatants, add ammonium sulfate to 20%
saturation according to Table 9.1 [3] and perform as described
above. The protein precipitates obtained at various saturation of
ammonium sulfate are dissolved in small volume of appropriate
buffer, and dialyzed against the same buffer. After overnight
dialysis, dialysates are collected, and their enzyme activity and
protein concentration are measured. The fractions containing
the highest total activity are used for further purifications.
9.2.1.2 Precipitation with organic solvents

Addition of water-miscible organic solvent such as acetone or
ethanol to the extract obtained above leads to the precipitation
of protein. The principle of the method is similar to that for
ammonium sulfate precipitation. That is, water molecules on the
surface of protein are decreased by the addition of the organic
solvent; thus, protein molecules interact with each other to
precipitate. Acetone is a good solvent for the protein precipitation.
Usually acetone cooled to –20°C is added slowly to the extracts
of protein. Organic solvents are mostly a denaturant of proteins.
However, by adding cold acetone, the denaturation of proteins are
prevented to a small extent under low temperature.
9.2.2 Column Chromatography

Column chromatography (adsorption) was invented by M. Tswett
in 1906 [4]. He separated plant pigments using calcium carbonate
as a stationary phase (carrier, or media) in a column, and carbon
disulfide (or petroleum ether) as the mobile phase. He was the
first to use the terms chromatogram and chromatographic method.
Figure 9.1 shows the schematic view of the chromatography.
Here, most commonly used methods: ion exchange, gel filtration,
and affinity chromatographies are mentioned. Other methods are
described in [1, 2]. The chromatography is grouped into several
methods by the difference in the interaction between the stationary
and the mobile phases. Before mentioning the methods, materials
of solid supports for the stationary phase are described.
Figure 9.1 Schematic view of column chromatography. A mixture of

proteins A, B, and C were applied on the top of the column, and
washed with an elution buffer. The eluates were collected by
the fraction collector. Proteins were eluted in the order of A, B,
and C. From Suzuki, H. World of Enzyme (in Japanese) [6].
Cellulose: polymer of glucose with b-1,4 linkage, having a linear

structure.
Agarose: polymer of d-galactose and 3,5-anhydro-l-galacto-
pyranose linked by a-1,3 and b-1,4 glycosidic bonds. Extracted from
seaweeds.
Dextran: polymer of glucose composed mainly of a-1,6
glycosidic bonds. It is synthesized from sucrose by certain lactic
acid bacteria.
Polyacrylamide: polymer of acrylamide. It is usually cross-linked
with N,N¢-methylenebisacrylamide.
9.2.2.1 Ion exchangers

This is the method to separate proteins using the difference in their
affinity to the ion exchangers in a column. Most commonly used
exchangers are described.
Anion exchanger: Diethylaminoethyl (DEAE)-group is an ion
exchanger. Cellulose, agarose, and dextran contain hydroxyl groups,
which form a linkage with the DEAE group,
Solid support–O–CH2CH2–N+(CH2CH3)2
Thus, the DEAE group is positively charged (pKa 11.5) under neutral
conditions and binds with the negatively charged ion in the buffer
such as Cl–. When proteins are applied on the top of the column, Cl¯
exchanges with the negatively charged portion of the proteins. The
strength of the binding depends on the negative charges of proteins;
thus, the column can separate proteins.
Cation exchangers: A carboxymethyl (CM) group is a cation
exchanger. Hydroxyl group of carbohydrate-based supports forms a
linkage with CM group,
Solid support–O–CH2COO–
Thus, CM group is negatively charged (pKa 4.7) under neutral
conditions, and interacts with positively charged proteins.
9.2.2.2 Gel filtration

The stationary phase of column is gel beads composed of
polysaccharide or polyacrylamide. Gel beads with various pore
sizes are available commercially. The principle of separation of
protein is roughly explained. When different size of proteins are
applied on the top of the column packed with gel beads, and eluted
with an appropriated buffer, the large-sized proteins mostly run
through among the column beads, but small-sized proteins enter
into the pore of the gel. Thus, larger proteins have shorter path in
the column, but smaller proteins longer path, leading to the
separation of proteins on the basis of molecular weight (MW).
The longer column with short diameter will enhance the good
separation of proteins. Various pore-sized gel beads are commercially
available. We can select the gel beads from the expected MW
of enzyme of interest, and use the column for the purification
and analysis of enzymes.
Gel filtration of proteins can determine MW of protein under
native conditions (Fig. 9.2). Therefore, for the oligomeric proteins,
we can estimate the subunit composition of the proteins, if we
perform the analysis of sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) described in the later section.
For example, we determined MW of a protein to be 80,000 under
native conditions, but the protein showed only one band of MW
of 40,000 by SDS-PAGE. The results suggest that the protein is a
dimer of a subunit with MW of 40,000.
Figure 9.2 Estimation of molecular weight of a sample protein of unknown

molecular weight (MW) by gel filtration chromatography. MW
is expressed as kilo unit, and the elution volume is appropriate
unit. From the elution volume of sample protein (arrow head),
MW of the protein is estimated from the log (MW) value.
9.2.2.3 Affinity chromatography

This method utilizes the fact that enzyme binds specifically with
specific ligands. The affinity is quite specific; thus, we can purify
the enzyme of interest in the mixture of proteins. Porous supports
are agarose, dextran, and polyacrylamide beads. Various ligands are
conceivable, and affinity columns are available commercially. Some
examples are shown below.
Immunoglobin G (IgG): IgG is not an enzyme, but good for
understanding the principle affinity chromatography. Protein A is
50 kD surface protein found in the cell of the bacterium
Staphylococcus aureus, and specifically binds with IgG. The
activated supports such as agarose and dextran beads couple
with amine groups of protein A, and the resulting beads (such
as protein A sepharose) can specifically bind with IgG. Apply the
solution containing IgG, and wash the column extensively with a
buffer solution to remove unbound proteins. IgG is eluted with
the appropriate solution.
Recombinant enzymes (proteins): The most common method
to prepare the enzyme of interest is to get the recombinant enzyme
in an appropriate expression system as described in Chapter 7.
When we express the enzyme with a tag at its N- or C-terminal
side, the extract obtained from the expressed cells are applied onto
the affinity column that specifically binds with the tag, but the
other proteins in the extracts cannot bind with the column; thus,
we can purify the enzyme of interest. For the purification of the
recombinant enzymes, immobilized metal affinity chromatography
is commonly used [5]. The column uses the fact that transition
metal ions such as Zn2+, Cu2+, Ni2+, and Co2+ bind to His and Cys in
aqueous solutions. Ni2+ ion binds to the iminodiacetic acid bound
covalently with the polysaccharide-based beads such as agarose
or dextran (Fig. 9.3). When the 6His-tagged recombinant enzyme is
applied onto the column, the enzyme binds the column specifically,
but other proteins without 6His tag do not bind with the column.
After washing the column with buffer, the enzyme is eluted by
buffer containing imidazole, leading to one-step purification of
enzymes (proteins).
Figure 9.3 Binding site structure of Ni-affinity column. Imino diacetic

acid to fix Ni2+ ion is shown. Three atoms are coordinated with
the metal ion (tridentate). The imidazole N of His6 region of
the His tagged protein coordinate with the Ni2+, thus the
His-tagged protein is bound to the column. To increase the
affinity of Ni2+ ion to the solid support, nitrotriacetic acid
(tetradentate), and tris-carboxymethyl ethylene diamine
(pentadentate) are used as the chelating ligand, the columns
with these ligands are commercially available.
9.3 Purity Analysis of Enzyme

To study on the enzyme from kinetic and structural basis, the enzyme
of interest should be pure. Standard and commonly used methods
are described. The basis of the method is electrophoresis.
Purity Analysis of Enzyme 179
9.3.1 Electrophoresis
Electrophoresis is the motion of charged particles under the
electric field. The positively charged particles moves toward the
cathode, and the negatively charged particles to the anode. Proteins
(enzymes) are composed of amino acid residues, so different
proteins are charged differently. Using the properties, various types
of electrophoresis apparatus are invented. Figure 9.4 shows an
example of slab gel electrophoresis apparatus.
Figure 9.4 Overview of slab gel electrophoresis apparatus. From Suzuki,

H. World of Enzyme (in Japanese) [6].
9.3.2 Sodium Dodecyl Sulfate Polyacrylamide Gel

Electrophoresis
SDS is an amphipathic molecule, and binds with proteins
approximately 1 molecule per 2 amino acid residues. The SDS-
bound proteins are denaturated to form the stick-like structure, and
negatively charged. These proteins move toward the anode under
an electric charge. SDS-PAGE is the method that carries out the
electrophoresis using the polyacrylamide gel as the solid support
(Fig. 9.5). The proteins enter the gel matrix, and move to anode.
Proteins with small size (molecular weight) move faster than those
with large size. Thus, proteins are separated on the basis of their
molecular weight.
Figure 9.5 Formation of polyacrylamide from acrylamide and N,N¢-

methylenebisacrylamide (cross-linking reagent) in the
presence of ammonium persulfate as the initiator and
N,N,N¢,N¢-tetramethylethylendiamine as the catalyst.
(a) (b)
Figure 9.6 Analysis of protein by SDS-PAGE. (a) Lane 1, proteins of MW

standard; lane 2, sample protein. (b) Log (MW) vs. protein
mobility (arbitrary unit). From the mobility of sample
protein(arrow head), MW of the protein is estimated. MW is
expressed as in (a).
9.3.3 Isoelectric Focusing

This is a method for separating molecules by differences in their
isoelectric point (pI). Isoelectric point means the pH of the solution
that the net electric charge of the solute is zero. Proteins are
composed of amino acid residues, so proteins have different
Purity Analysis of Enzyme 181
electric charge from one protein to other. When a mixture of

proteins in the pH gradient of the solid matrix (usually agarose
gel) is subjected to an electric field, proteins migrate toward the
anode or cathode depending on their net charges, and stop to
migrate at the local region with the pH of the pI of protein. This
phenomenon is called isoelectric focusing. For example, a protein
in a pH region below its pI will be positively charged, so the protein
migrates toward the cathode.
The pH gradient is prepared by applying the electric field
to the mixture of ampholite in the matrix gel. Ampholite means a
twitter ionic compound such as amino acid. For electric focusing
experiments, aliphatic oligoaminocarboxylic acid (Fig. 9.7) is
ampholite and commercially available, such as Pharmalyte from GE
Healthcare Bio-Sciences.
Figure 9.7 Structure of ampholite for isoelectric focusing.
Here the method to prepare the pH gradient gel is briefly

described. Prepare appropriate concentration of agarose gel
solution containing ampholite of pH regions of interest, pour the
agarose gel solution into a glass tube. Leave the tube with agarose
in the cold room (usually 5°C) to form the gel. After the gel is
formed, protein samples are applied on the top of the gel
tube. Then, the electric charge is applied between the top and
bottom of the tube. The top is cathode filled with 200 mM NaOH
solution, and the bottom is anode with 10 mM phosphoric acid.
When the electric field is applied, the pH gradient is formed, and
proteins migrate toward anode or cathode depending on their
electric charge of proteins. The migration of proteins will stop at
the pH of the protein’s pI.
Two-dimensional electrophoresis is the method to analyze
proteins more strictly. One-dimensional electrophoresis, for
example SDS-PAGE, could easily determine the purity of enzyme.
However, if there are two proteins having same molecular weight,
but their pI values are different each other. SDS-PAGE could
not conclude whether the enzyme is pure. In these cases, two-
dimensional gel electrophoresis is useful. Practically, after

proteins are separated on the basis of their pI, the gel tube is
removed from the glass tube. The gel is set on the top of the PAGE
slab gel, and the SDS-PAGE is performed (Fig. 9.8). This type of
experiments is used for proteomic studies.
Figure 9.8 Two-dimensional gel electrophoresis. First, perform the

isoelectric focusing gel electrophoresis of protein samples.
Cathode is filled with an alkaline solution, such as 200 mM
NaOH, and the anode an acid solution, such as 10 mM
phosphoric acid. After the isoelectric focusing, the gel is put on
the top of the gel for SDS-PAGE. After electrophoresis, proteins
in the gel is stained with a protein staining reagent, such as
Coomassie brilliant blue.
Problems
1. A DEAE-cellulose equilibrated with a buffer (pH 7.0) was
packed in a glass column. A solution containing bovine
serum albumin (pI = 4.7), human hemoglobin (pI 6.5), and
bovine cytochrome c (pI = 10) in the buffer (pH 7.0) was
applied on the top of the column. Then, the column was washed
with the same buffer. A protein is eluted from the column.
After complete elution of the protein, the column was washed
step-wise with the buffer containing increasing concentrations
of NaCl. Answer the order of the elution of these three
proteins.
2. In question 1, the concentration of NaCl was increased to wash
the column. Why?
References 183
3. Catalase is a protective enzyme by the decomposition of

hydrogen peroxide. A SDS-PAGE analysis of the enzyme shows
a single polypeptide with approximate MW of 60,000. Under
native conditions, the MW of the enzyme is approximately
250,000. Determine the number of subunits in the native
catalase.
References
1. Scopes, R. K. (1988). Protein Purification. Principles and Practice, 2nd

ed. (Springer-Verlag, USA).
2. Methods Enzymol. (Elsevier, Holland). A series of scientific publications
focused primarily on research methods in biochemistry. First published
1955 from Academic Press.
3. Green, A. A., and Hughes, W. L. (1955). Protein fractionation on the
basis of solubility in aqueous solutions of salts and organic solvents.
Methods Enzymol., 1, 67–90.
4. Tswett, M. (1906). Physikalich-Chemische Studien über das Chlorophyll.
Die Adsorption. Berichte der Deutschen botanischen Gesellschaft., 24,
384–393.
5. Block, H., Maertens, B., Spriestersbach, A., Brinker, N., Kubicek, J.,
Fabis, R., Labahn, J., and Schäfer F. (2009). Immobilized-metal affinity
chromatography (IMAC): A review. Methods Enzymol., 463, 439–473.
6. Suzuki, H. (1996). World of Enzyme (Sangyo Tosho Co. Tokyo, Japan)
(in Japanese).
Chapter 10
A Case Study: l-Phenylalanine Oxidase

(Deaminating and Decarboxylating)
This chapter describes an example of enzyme research. In

the previous chapters, examples are shown for the better
understanding of the respective topics. The enzyme, l-phenylalanine
oxidase (deaminating and decarboxylating) (PAO), is unique in
that it catalyzes both oxidative deamination and oxygenative
decarboxylation of l-Phe, l-Tyr, and l-Met, and highly specific
to l-Phe. Moreover, the structural studies of PAO revealed a
novel type of activation, that is, PAO is expressed as pro-enzyme
(proPAO), and the removal of the prosequence which occupies
the substrate channel results in the activation of enzyme.
10.1 Introduction
l-Phe oxidase (deaminating and decarboxylating) was purified
from Pseudomonas sp. P-501 by H. Koyama of Noda Institute for
Scientific Research (Japan) in 1983 [1]. Dr. Koyama and we started
to collaborate on the kinetic study of the enzyme. We have studied
on the enzyme kinetically and structurally since then. So it is worth
to look back at the works here.

Haruo Suzuki
www.panstanford.com
186 A Case Study
10.2 Preparation of PAO

The methods initially used by Koyama are described. For the
crystallographic research, elaborate instruments for purification
were used, but not mentioned in this chapter.
10.2.1 Preparation of the Cell Extracts

Koyama intended to get a highly specific enzyme for l-Phe, since
it may be useful to determine the amount of l-Phe in the clinical
laboratory. In this chapter, the expression of “L”-isomer is omitted
for simplicity. Koyama isolated the bacterium, Pseudomonas sp.
P-501, from soil by an enrichment-culture technique with Phe as
the sole carbon and nitrogen sources, and taxonomically
characterized as a strain of genus Pseudomonas according to
Bergey’s Manual [1]. Cells were grown aerobically for 18 h at 30°C
in a medium as described [1], and harvested by centrifugation at
the end of the exponential phase of growth. The harvested cells
were treated with a surface-active reagent, Triton X-100 (0.1%) in
20 mM potassium phosphate buffer. The cell debris was removed
by centrifugation, and the supernatants were used for the
purification of PAO.
10.2.2 Purification of PAO by Column

Chromatographies
The cell extracts were directly applied to the top of the column of
DEAE cellulose, which was pre-equilibrated with 20 mM potassium
phosphate buffer (pH 7.0). PAO was adsorbed to the column, which
was then washed with the same buffer containing 50 mM NaCl.
PAO was eluted from the column with the same buffer containing
250 mM NaCl. The proteins in the eluates were concentrated by
the addition of ammonium sulfate. The concentrated proteins in the
buffer were applied on the top of the column of phenyl-sepharose,
which is pre-equilibrated with the buffer containing ammonium
sulfate (5% saturation). The column was washed with the buffer
containing ammonium sulfate (5% saturation), and the PAO is eluted
with the buffer containing decreasing concentration of ammonium
Preparation of PAO 187
sulfate. The active fractions were collected and concentrated by

the addition of ammonium sulfate. The phenyl sepharose column
chromatography is a kind of hydrophobic column chromatography,
which was not mentioned in Chapter 9. The hydrophobic interaction
is increased in high ionic strength. Therefore, the hydrophobic
ligands, such as phenyl groups, interact with the hydrophobic
portions of protein, but the interaction will decrease when ionic
strength is lowered. Ammonium sulfate is usually used to increase
the ionic strength of the solution, since it is mild reagent for
proteins. The active fractions were collected and concentrated by
the addition of ammonium sulfate. The concentrated proteins were
applied to a column of Bio-Gel A-0.5 m, which is the gel bead for
the size exclusion chromatography from Bio-Rad Inc. The
fractionation range of the gel beads is 10–100 kDa. PAO prepared
was shown to be purified as a single band by a gel electrophoresis.
From 1.95 g protein of the crude extract, 2.9 mg of purified PAO was
obtained [1]. Molecular properties of PAO are shown in Table 10.1
[2, 3]. PAO is a flavoprotein containing two FADs per one molecule
of enzyme. The enzyme is heat stable and has broad optimum pH of
activity. The pI 4.8 of PAO explains the adsorption of enzyme to the
anion exchange column (DEAE cellulose) at pH 7.0.
Table 10.1 Molecular properties of PAO
MW 140,000
Subunit composition single
Subunit MW 68,000
Isoelectric point 4.8
FAD content 2
Optimum pH 6~9
Heat stability ~70°C
Note: Subunit composition was determined by SDS-PAGE. Molecular weight (MW) of

PAO was determined by a gel chromatography. Heat stability was tested by assaying
the remaining activity after 10 min incubation of enzyme in 50 mM potassium
phosphate buffer at pH 7.0 at room temperature to 70°C (summarized from refs. 2
and 3). The enzyme is now known to be composed of a2 and b2 [4].
188 A Case Study
10.3 Catalytic Properties of PAO

10.3.1 Stoichiometry of the Reaction Catalyzed by PAO
Stoichiometry means “the quantitative relationship among
reactants and products in chemical reactions.” When Phe was used
as substrate, β-phenylpyruvate, α-phenylacetamide, CO2, and NH3
were formed with the consumption of molecular oxygen.
The amounts of Phe and oxygen consumed and those of
β-phenylpyruvate, α-phenylacetamide, CO2, and NH3 formed showed
that PAO catalyzes the following two reactions: the oxidative
deamination (20%) and oxygenative decarboxylation (80%).
C6H5CH2CH(NH3+ )COO– + O2 + H2O C6H5CH2COCOO– + NH+4 + H2O2 (10.1)
C6H5CH2CH(NH3+ )COO– +O2 C6H5CH2CONH2 + CO2 + H2O (10.2)
The oxygenation reaction of PAO was confirmed that an 18O atom

incorporates into α-phenylacetamide in the presence of 18O2 [3].
In other proteinogenic amino acids studied, Tyr and Met were 4 and
100% oxidized, respectively. Therefore, Phe and Tyr are to be called
the oxygenase substrates, and Met is called the oxidase substrate.
PAO has the highest activity for Phe, and 44% for Tyr, 19% for
Met, 9% for Trp and 9% for Ala as compared with activities of
Phe. Non-proteinogenic analogue of Phe, β-2-thienylalanine (βTA)
has relatively lower activity (40%) than Phe: βTA is Phe antagonist
used in Guthrie test for phenyketonuria (PKU).
β-2-thienylalanine
10.3.2 Overall Reaction Kinetics

Using Phe, Tyr, βTA, and Met as substrate, the rate of oxygen uptake
was measured at various concentrations of substrate amino acid
and oxygen. A reciprocal plot between the rate of oxygen uptake
and substrate or oxygen concentrations gave parallel lines.
Catalytic Properties of PAO 189

(b) [O2] mM [O2], mM
(a) 0.53 (c) (d)
0.008 0.008 0.18
0.77 0.050 0.050 0.27

1.04
0.48
e0/v, s
1.13
0.004 0.004
0.025 0.025
1.02
[ßTA] ĺ
[O2] ĺ [Met] ĺ [O2] ĺ
0 0
0 1 2 0 10 20 0 0
0 2 5 0 2 2 3
1/[O2], mM-1 1 [ßTA], mM-1 1/[O2], mM-1
1/[Met], mM-1
Figure 10.1 Effect of concentrations of substrate amino acids and oxygen

on the rate (v/e0) of the PAO-catalyzed reaction at pH 7.0
and 25°C. (a, b) Effect of various concentrations of βTA and
oxygen on the rate of PAO-catalyzed reaction. (c, d) Effect
of various concentrations of Met and oxygen. Adapted from
Koyama and Suzuki [5].
Figure 10.1 shows the results obtained with βTA and Met [5]. The
data clearly show a typical Ping-Pong Bi–Bi mechanism as described
in Chapter 3:
1 k
k2 slow

Eox +S   E S  Ered Im  Ered + P1 (10.3)
k  ox –1
k3 k

 Ered ImO2 
Ered Im+O2 (+H2O) 

4
Eox + P1 + H2O2 + NH+4
k–3
(10.4)
3 k
k4

Ered Im+O2   Ered ImO2 
 Eox +P2 +CO2 +H2O (10.5)
k–3
k
1 k2 1 slowk2 k slow

Eox +S   E ES 
where and
+S Ek red
 Im
represent
Eox
S  EE
the +PIm
oxidized
1  and Ereduced
red + P1 forms of
k–1  ox ox 
–1
red
red
the enzyme-bound FAD cofactor, respectively. Im represents the
imino acid corresponding to the amino acid substrate. Equation
10.4 composes the oxidative deamination reaction (Met), and
Eq. 10.5 the oxygenative decarboxylation reaction (Phe, Tyr, βTA).
k1 k2 slow
EThe 
ox +S imino
 acid
E Sin the Ered·Im
Im  Ered + Pwas
intermediate identified by a
k–1  ox 1
resonance Raman study of the purple intermediate formed with Phe
[6]. The substrate used follows either Eq. 10.4 or 10.5, therefore,
rate constants were assumed to be identical in each steps in
Eqs. 10.4 and 10.5 for simplicity. From the mechanism, the rate
of overall reaction (v) can be expressed as
e0 e  Ks  e KO 2
= 0 1 + m  + 0 m (10.6)
v V  [S]  V [O2 ]

190 A Case Study
V kk k4 (k–1 + k2 ) k (k + k )
s
= 2 4 = kcat , K m = , K mO2 = 2 –3 4
e0 k2 + k4 k1 (k2 + k4 ) k3 (k2 + k4 )

where e0 represents the total concentration of enzyme. The
enzyme concentration is expressed as that of the FAD content. V
represents the maximum rate at a given concentration of enzyme
and at the infinite concentrations of both substrate and oxygen,
k (k + kk2 2)(k–3 + k4 )
and K ms =and4 K–1mO2 =the ,
concentration of substrate and oxygen to give
k (k2 + k4k)3 (k2 + k4 )
the rate of1 V/2e 0, respectively. The kinetic parameters obtained
are shown in Table 10.2. The parameters show that though the
k2 (k–3 + k4 )
kcat values of PAO are very high, K mO2 = is very large as compared with
k ( k + k ) k3 (k2 + k4 )
K ms . =This
4 –1 ,
fact 2explains the relatively low activity under standard
k1 (k2 + k4 )
conditions (25°C and 1 atm), where the oxygen concentration is
0.25 mM.
Table 10.2 Kinetic parameters of the steady state of reaction catalyzed by

PAO at pH 7.0 and at 25°C
s o
Amino acid kcat, s–1 K m , mM K m2 , mM
Phe 1850 0.1 3.15
βTA 800 0.07 1.84
Met 286 2.2 1.26
Tyr 3570 4 6.78
Source: Adapted from Koyama and Suzuki [5].
10.3.3 Determination of Kinetic Constants

Table 10.2 shows that PAO has the high kcat values. This indicates
that the rates of the step from Eox·S to Ered·Im and from Ered·Im·O2
to Eox are large (Eqs. 10.3, 10.4, and 10.5). Figure 10.2 shows the
spectral change of PAO by the addition of Phe or Met. The enzyme
shows a typical spectrum of flavoproteins. By the addition of
substrates, the color of enzyme changes yellow to purple. The
purple-colored species are stable under anaerobic conditions. To
determine the rate of reduction of the FAD cofactor, the stopped-
flow method was used (see Chapter 5). The kcat value for Phe is
very large (Table 10.2), so the decrease of absorbance at 465 nm
was complete during the dead time of the stopped flow apparatus
(2.3 ms) under anaerobic conditions. Therefore Met was used as
substrate for the stopped flow studies, since kcat value of Met is
lower than that of Phe (Table 10.2).
0.3 0.06
A B
a a
Absorbance
0.2 0.04
0.1 b 0.02
b
0 0
340 430 520 610 700 340 430 520 610 700
Wavelength, nm Wavelength, nm

Figure 10.2 The spectral change of the oxidized form of enzyme (Eox, a)
to the purple intermediate (Ered·Im, b) by the addition of
13 mM Phe (A) and 8 mM Met (B) under aerobic conditions.
PAO concentration was 20 μM (A) and 4.4 μM (B). As Kom 2 is
high, it is practically anaerobic with sufficient concentrations
of the amino acid. Reproduced from Ohta et al. [7].
When PAO was mixed with Met using a stopped-flow apparatus

under anaerobic conditions, the absorbance at 465 nm (A465) was
analyzed on the basis of the first-order-reaction, fitting Eq. 10.7:
A465 = C exp(–kobst )+ b (10.7)
where C is a constant related to the initial absorbance change, kobs

the observed rate constant for the flavin reduction, and b an offset
value to account for non-zero base line. The rate is automatically
determined by the software equipped with the apparatus. A kobs
value is expressed as a function of [S],
k2
kobs =
K (10.8)
1+ s
[S]
where Ks represents a dissociation constant of the EoxS complex.

A plot of 1/kobs vs. 1/[Met] gave linear line, leading to determine
the rate of reduction of the FAD cofactor. At 10°C and pH 7.0, k2
and Ks were determined to be 420 s–1 and 420 μM, respectively.
192 A Case Study
10.3.4 Hydrogen Quantum Tunneling in the

PAO-Catalyzed Reaction
10.3.4.1 Hydrogen quantum tunneling (hydrogen tunneling)
Hydrogen (hydrogen, proton, or hydride: H) quantum tunneling is

described to some extent in Chapter 4. Before focusing the
H-tunneling in the PAO-catalyzed reaction, I briefly mention
H-tunneling.
Matter has a nature of both particle and wave. This is usually
called “wave-particle duality.” For example, light is treated as not
only “particle,” but also “wave.” Luise de Broglie proposed a so-called
de Broglie wavelength, which is inversely proportional to the square
root of the mass of the particle. Therefore, the wavelength is longer
with lighter particles, and quantum tunneling is more likely to be
observed with lighter particles. In biological systems, the electron
quantum tunneling is widely known. The second light particle is
hydrogen atom, and the hydrogen quantum tunneling was observed
with yeast alcohol dehydrogenase by Klinman and coworkers [8].
Since then, H tunneling was reported for various enzymes [9–11],
and seems to be a common mechanism of the enzymatic hydrogen
transfer reactions.
The energy change of the carbon-hydrogen bond cleavage is
shown in Fig. 10.3 [12]. In the figure, protium (H) and deuterium
(D) transfers are shown. The quantum mechanics tells us that the
C–H and C–D bonds are vibrating even in the ground state. The
bond energy can be calculated from the accumulated data of the
infrared spectroscopy. Assuming that the C–H and C–D vibrational
frequencies are 2900 and 2100 cm–1. Then, the zero point energies
are calculated to be 17.3 and 12.5 kJ/mol, and the primary kinetic
isotope effect (kH/kD) is a function of the difference in the zero point
energy of these bonds (4.8 kJ/mol). Therefore, kH/kD is calculated
to be ~7 according to the Arrhenius equation (Eq. 1.2 in Chapter 1).
This value has been an indicator whether the H transfer proceeds
by quantum tunneling or the over the barrier. That is, the higher
kinetic isotope (>7) indicates “quantum mechanical tunneling.”
However, it has been pointed out that the higher KIE alone is not a
reliable indicator of H-tunneling [9].
(a) (b)
Figure 10.3 Reaction coordinates showing the deuterium isotope effect

of the carbon-hydrogen bond cleavage reaction. (a) The
difference in the activation energy (Ea) between the C-H and
C-D bonds is derived from that in the zero point energies. (b)
The difference in the activation energy derives also from the
quantum mechanical tunneling through the reaction barrier
width. Notice that the deuterium and protium particles tunnel
different barrier widths. Adapted from Park, H., Girdaukas,
G. G., and Northrop, D. B. (2006) Effect of pressure on a
heavy-atom isotope effect of yeast alcohol dehydrogenase.
J. Am. Chem. Soc., 128, 1868–1872, copyright(2006), American
Chemical Society.
Saunders demonstrated in 1985 from the analyses of elimination

reactions [13] that the relation of H/T to D/T kinetic isotope effects
deviates significantly from
kH/kT = (kD/kT)3.26 (10.9)
when the quantum mechanical tunneling is significant. Klinman

et al. [8] measured the rate of dehydrogenation of the isotope-
labeled benzylalcohol by yeast alcohol dehydrogenase. They used
the protium-, deuterium-, and tritium-labeled substrates, and found
(kH/kT)obs > (kD/kT)3.26 = (kH/kT)cal (10.10)
where kH/kT and kD/kT are the H/T and D/T kinetic isotope effects,
respectively. Here, subscript obs and cal represent observed and
194 A Case Study
calculated values, respectively. The result clearly shows that the

hydrogen tunneling contributes significantly in the C–H bond
cleavage by alcohol dehydrogenase. Later, Klinman et al. [14]
proposed a criteria for the determination if or not the H-transfer
occurs with quantum tunneling using the Arrhenius plot,
–Ea (10.11)
lnk = + ln A
RT
In Fig. 10.4 [9, 14], the region I follows the Arrhenius rate law,
showing large DH≠, and AH/AD ≈ 1. Here A is an Arrhenius pre-
exponential factor. In the region II, inflated value of kinetic isotope
effect and AH/AD < 1 will be given, and the behavior reflects a greater
tunneling. In the region III, extensive tunneling occurs, but AH/AD
is hard to predict. In the region IV, nearly activationless tunneling
and KIE ≈ AH/AD.
(a) (b)
Figure 10.4 A general diagram of the rate (k) of hydrogen transfer reaction
with temperature. The region I, classical behavior of the over
the barrier, the regions II and III, quantum tunneling, and the
region IV, ground-state quantum tunneling. For Arrhenius plot,
read the ordinate in (b) as ln k [14] (See text). Reproduced
with permission from Scrutton, N. S., Basran, J., and Sutcliffe,
M. J. (1999). New insights into enzyme catalysis. Ground
state tunneling driven by protein dynamics. Eur. J. Biochem.,
264, 666–671, copyright (1999) John Wiley & Sons. (b)
Adapted with permission from Jonsson, T., Glickman, M. H.,
Sun, S., and Klinman, J. P. (1996) Experimental evidence for
extensive tunneling of hydrogen in the lipoxygenase reaction:
Implications for enzyme catalysis. J. Am. Chem. Soc., 118,
10319–10320, copyright (1996), American Chemical Society.
The Arrhenius plot is phenomenological, so Scrutton et al.

proposed the method that uses the Eyring plot in place of Arrhenius
plot [11]. That is, the temperature dependence of a unimolecular

reaction rate is expressed in Eq. 10.12:
kBT  –DG  kBT  – DH    DS  

kf = exp = exp exp  (10.12)
h  RT  h  RT   R 

divide both sides by T, then logarithm of both sides gives the Eyring
plot:
 k T  DS  DH 
ln(k f /T )= ln B + – (10.13)
 h  R RT
They analyzed methylamine dehydrogenase (MADH)–catalyzed

reaction at 5 to 40°C using the deuterated and protiated methylamine
as substrate [11]. The KIE was independent on temperature and
almost equal to AH /AD , indicating activationless ground state
tunneling. Here, the prime is used to discriminate the value
obtained by the Eyring plot from the A value obtained by the
Arrhenius plot. However, DH≠H and DH≠D were almost the same value
of 45 kJ/mol, that is, not zero. The results are not consistent with
the criteria, the activationless H-transfer, indicating the limit of
the criteria shown in Fig. 10.4. However, the rate data analysis
shown in Fig. 10.4 is valuable as the initial consideration
of H-tunneling study.
10.3.4.2 Hydrogen tunneling in the PAO-catalyzed reaction

As described in Table 10.2, the PAO-catalyzed reaction is very rapid
process when Phe is used as substrate. Therefore, we selected
Met as substrate, since it has a relatively low activity in the amino
acids studied. The maximum rate of reduction was 420 s–1 and the
dissociation constant of Met from the Eox · Met complex was 420 μM
at 10°C. Therefore, the rate of reduction was determined with
8 mM Met at 10–30°C. Figure 10.5 shows the Eyring plot of the
data obtained. From the Figure, the difference in the enthalpy
change of activation for protium vs. deuterium was calculated to be
–0.2 kJ/mol. The KIE is independent on temperature, and its
value of 5.4 agrees with the AH /AD , value of 5.2. These agree
with Klinman’s criteria of the activationless ground state H-
tunneling (the region IV in Fig. 10.4). However, like methylamine
196 A Case Study
dehydrogenase as described above, the activation enthalpy is not

zero, meaning that the active site residue(s) in the EoxS complex
of PAO are in dynamic motion and reorganize temperature-
dependently the conformation of the active site to be compatible
with H-transfer. In the presence of 30% glycerol, fluctuations
(dynamic motions) of the PAO active site must be hindered, and
the higher activation energies must consequently be required to
induce an active site conformation compatible with H-tunneling
as compared with a conformation without glycerol. Thus, less
fluctuation attenuates the H-tunneling and leads to decreased KIE
(4.1).
Figure 10.5 Temperature dependence on the rate (kred) of the reductive

half reaction of PAO with l-Met. (a) [Cα-H]-Met (solid circles)
or [Cα-D]-Met (solid triangles) was used as substrate in the
absence of glycerol. DH≠H = 23.5 kJ/mol, DH≠D = 23.7 kJ/mol
and AH / AD = 5.2 were obtained from the plot. Inset, plot of
ln(KIE) vs. 1/T. (b) Experiments similar to (a) were performed
in the presence of 30% glycerol. DH≠H = 33.8 kJ/mol, DH≠D =
31.2 kJ/mol and AH / AD = 5.2 were obtained from the plot. The
concentration of Met was 8 mM. Inset: plot of ln (KIE) vs. 1/T.
Adapted from Ohta et al. [7].
10.4 Structural Properties of PAO

The previous sections described on the catalytic nature of PAO.
This section focuses on PAO as a molecule.
10.4.1 Nucleotide and Its Deduced Amino Sequences of

PAO Gene and Its Expression
PAO was initially known to be composed of two identical subunits,
but later it was shown to be composed of non-identical two subunits,
Structural Properties of PAO 197
a and b [4]. For the preparation of enzyme, autolysis is required and

the yield of the enzyme was known to increase by the addition of
Pronase. These facts suggest that the enzyme must be expressed
as a pro-enzyme, which is activated by proteolysis. Therefore, the
DNA sequence encoding PAO from Pseud. sp. 501 was determined.
Comparison of the deduced amino acid sequence with the partial
amino acid sequences previously obtained showed that PAO is
expressed as pro-enzyme, proPAO, and proteolytically activated.
This was confirmed by the fact that proPAO expressed in E. coli is
activated well with the mixture of Pronase and trypsin (Fig. 10.6)
[15, 16]. ProPAO is composed of two identical subunits, and has
the prosequence with 14 residues length. ProPAO does not have
catalytic activity, but treatments of proPAO with various proteases
change it to the active form. Highest activity was obtained by the
treatment of proPAO with the mixture of Pronase and trypsin.
(a)
(b)
(c)
Figure 10.6 Amino acid sequences of proPAO (a), PAOpt (b), and the
native PAO (c). Though proPAO is a homodimeric protein,
only monomer is shown. Only partial sequences are shown.
Numbers above the sequence represent the residue number
according to the prosequence. The cleavage positions shown
by arrow heads are different between recombinant PAOpt and
the native enzyme. The LEH6 sequence of proPAO represents
the histidine tag added in the construction of the expression
plasmid in E. coli.
Thus, the activated enzyme was named as PAOpt. PAOpt has

the catalytic activity comparable with the native enzyme, so the
X-ray crystallographic analyses were performed with these
recombinant PAOs.
198 A Case Study
10.4.2 3D Structures of proPAO and PAOpt

The 3D structures of proPAO and PAOpt are shown in Fig. 10.7. In
the prosequence of the proPAO structure (Figs. 10.7a and 10.8a),
(a) (b)
Figure 10.7 Overall structure of PAO. A view of proPAO (a) and PAOpt
monomers (b). The prosequence, FAD-binding domain,
and substrate-binding domain are colored, magenta, blue,
and orange (proPAO) or cyan (PAOpt), respectively. (a) and
(b) are shown in the same orientation. This research was
originally published in Journal of Biological Chemistry. Ida, K.,
Kurabayashi, M., Suguro, M., Hiruma, Y., Hikima, T., Yamamoto,
M., and Suzuki, H. Structural basis of proteolytic activation of
l-phenylalanine oxidase from Pseudomonas sp. P-501. Journal of
Biological Chemistry. 2008. 283:16584–16590. © the American
Society for Biochemistry and Molecular Biology [16].
(a) (b)
Figure 10.8 Structure of substrate channel. Cross-section views of

funnels in proPAO (a) and PAOpt (b). The carbon atom in the
prosequence is colored magenta. The molecular surface view
of funnel spaces in proPAO (a) and PAOpt (b). A is shown in
the same orientation as B. SO4 and GOL represent sulfate ion
and glycerol, respectively, and are shown by a stick model.
This research was originally published in Journal of Biological
Chemistry. Ida, K., Kurabayashi, M., Suguro, M., Hiruma, Y.,
Hikima, T., Yamamoto, M., and Suzuki, H. Structural basis
of proteolytic activation of l-phenylalanine oxidase from
Pseudomonas sp. P-501. Journal of Biological Chemistry. 2008.
283:16584–16590. © the American Society for Biochemistry
and Molecular Biology [16].
Substrate Specificity and Reaction Specificity of PAO 199
Figure 10.9 Illustration of proteolytic activation of proPAO. The scissor

represents the protease. The prosequence is colored magenta.
FAD is colored yellow. proPAO is cut at two sites, and then the
funnel occupied by the prosequence is closed. This research
was originally published in Journal of Biological Chemistry.
Ida, K., Kurabayashi, M., Suguro, M., Hiruma, Y., Hikima, T.,
Yamamoto, M., and Suzuki, H. Structural basis of proteolytic
activation of l-phenylalanine oxidase from Pseudomonas sp. P-
501. Journal of Biological Chemistry. 2008. 283:16584–16590.
© the American Society for Biochemistry and Molecular
Biology [16].
the peptide from Gly1 to Ile6 occupies the funnel-like structure,

which is composing the substrate channel. The structure clearly
shows the reason why proPAO has no catalytic activity. That is,
the cofactor FAD is buried inside the protein molecule, and the
N-terminal Gly of the prosequence attached to the flavin ring,
which is the site of action of the flavoprotein oxidoreductases.
On the other hand, in the PAOpt structure, the flavin ring is open
to the outside of protein through the funnel (Fig. 10.7b). These
changes are illustrated schematically (Fig. 10.9). Thus, the funnel
is the channel of the substrate. Interestingly, the funnel is closed
at the vestibule of the funnel by the hydrophobic residues, such
as Phe311, Met227, Leu330, Ile331, and Tys335. The role of these
residues will be considered in the next section.
10.5 Substrate Specificity and Reaction

Specificity of PAO
l-Amino acid oxidases usually are low substrate specificity, that
is, they oxidize most of proteinogenic amino acids. PAO and two
amino acid oxidases (l-Glu oxidase and l-Trp oxidase) are rare
cases with high substrate specificity. Moreover, PAO catalyzes
both oxidative deamination and oxygenative decarboxylation
(Fig. 10.10). Therefore, PAO is good target to study the substrate
and reaction specificities. Ida et al. studied these points from the
200 A Case Study
structural basis [16, 17]. Figure 10.11 shows the crystal of

recombinant PAOpt used in the X-ray crystallographic analyses.
The formation of the purple intermediate is confirmed by the
color change of crystal by soaking in the Phe solution.
Figure 10.10 Reaction mechanism of Pseudomonas l-Phe oxidase

(deaminating and decarboxylating). The enzyme contains two
FAD cofactors, but the scheme is expressed per FAD. PAM, PPV
and Im represent phenylacetamide, phenylpyruvate and imino
acid, respectively. Reproduced from Ida et al. [17].
First, let us consider the substrate specificity. We selected

LAO from Rhodococcus opcus (roLAO) as the enzyme being non-
specific to the amino acid side chain, and the active site structure
of PAO (pdb 3ayj) is compared with that of roLAO (pdb 2bj2)
(Fig. 10.12). The Cb and N atoms of Phe in both the PAOpt·Phe
complex and the roLAO·Phe complex interact with the aromatic
cage residues (F617, W660) via H-p bonds, but the bond lengths
in roLAO are longer than those of PAOpt. In addition to the cage
residues, the benzene ring of the substrate Phe is surrounded
by six hydrophobic residues in the PAOpt-Phe complex—F316,
L319, L330, Y335, W538, and L619—but in the roLAO-Phe complex,
the benzene ring appears to interact weakly with two hydrophobic
residues, F222 and I465. Therefore, the benzene ring of Phe is
more closely packed in the active site of PAOpt than in the active
site of roLAO. Thus, the active site pocket of roLAO can individually
accommodate the 20 proteinogenic amino acids. Moreover, the
trajectory of the substrate is gated by the hydrophobic residues
in the channel in PAOpt, but substrate freely diffuses through the
channel in roLAO. These facts show that PAOpt is highly specific
with respect to the side chains of the amino acids, while roLAO is
non-specific [16, 17].
Figure 10.11 Crystal of PAOpt. (a) The oxidized form. (b) The crystal
of the oxidized form (a) was soaked in the crystallization
buffer containing Phe. Thus, the crystal changed to purple.
Reproduced from Ida et al. [17].
(a)
(b)
Figure 10.12 Stereo view of the active site of PAO. (a), the PAO-Phe
complex (black) superimposed to that of roLAO (white). (b),
the PAO-Phe complex (black) superimposed to that of the
PAO-Met complex (white). The cage residues of PAO are
W660 and F617, and those of LAO are W467ro and W426ro.
Reproduced from Ida et al. [17].
202 A Case Study
Next, let us consider the reaction specificity of PAO. That is,

the enzyme catalyzes both the oxidative deamination and the
oxygenative decarboxylation. Phe is mainly oxygenated, and Met
mostly oxidized, therefore, the structure of the Ered-Im complex
may unveil the reason how one enzyme catalyzes two reactions
[17]. Unfortunately, however, Im in the complex was not stable
during the X-ray diffraction study as described in [17]. Moreover,
the step of oxidation is not well studied. Therefore, the reaction
specificity will be tentatively explained. The pKa of the amino group
of free Phe is 9.18, but the amino proton must be deprotonated
before dehydrogenation to accommodate the hydrophobic nature
of the active site. Probably, the proton must be released during the
migration from the outside of molecule to the active site.
Figure 10.13 Subunit of a and b chains of PAOpt (PAO: green) was matched
with that of human monoamine oxidase B (MAO: magenta).
FAD, cubic. The main chains are shown. PAO, pdb 3ayj; MAO,
pdb 1gos.
Accumulating data on the flavoprotein amine oxidases,

amine oxidases are grouped into two structural families [18],
the monoamine oxidase (MAO) and D-amino acid oxidase (DAO)
families. As Fig. 10.13 shows, the structure of PAO is well matched
with that of MAO, revealing that PAO is included in the MAO
structural family. The first step of the PAO-catalyzed reaction is
dehydrogenation of substrate. Structure of the PAOpt-Phe complex
suggests the direct hydride transfer of the α-H to the N5 atom
of isoalloxazine ring of FAD cofactor (Fig. 10.14). Although the

structures of the Ered-Im intermediate were not determined, water
molecule (Wa2 in Fig. 10.12) was observed both the complexes, and
the distance between the Ca atom of Met in the PAOpt-Met complex
is shorter than the corresponding distance for Phe.
Figure 10.14 Scheme of Phe dehydrogenation. Hydride ion is transferred

from the α-H of Phe. Reproduced from Ida et al. [17].
Figure 10.15 Scheme of oxidative deamination of Met by the PAO-catalyzed

reaction. Starting from the Ered·Im intermediate (I) to reform the
Eox form of enzyme (IV), and form 3-(methylthio) propionate,
ammonia, and hydrogen peroxide via II and III intermediates.
Adapted from Ida et al. [17].
204 A Case Study
Assuming that the imino acid intermediates are similarly

situated as shown in Fig. 10.12b, the imino form of Met reacts with
water easily to be hydrolyzed according to the oxidative mechanism
as shown in Fig. 10.15. Oxygen molecule probably enters to the
site of Wa1 through the channel different from the substrate
channel [16]. Although the presence of the peroxygenated form of
the reduced FAD cofactor is not identified, we assumed that the
Ered-OOH form is oxidized to reform the Eox form of FAD and
produce H2O2.
Figure 10.16 Scheme of oxygenative decarboxylation of Phe by the PAO-

catalyzed reaction. Starting from the Ered·Im intermediate (I)
to reform the Eox form of enzyme, phenylacetamide, carbon
dioxide, and water via II and III intermediates. Adapted from
Ida et al. [17].
In the case of Phe as substrate, reactions follow as shown in

Fig. 10.16. As the distance between Wa2 and Ca of Phe in the complex
is long. Thus, the Ca reacts with the peroxy group of the reduced
FAD rather than does with water molecule. Then decarboxylation
proceeds before the oxygenation to produce phenylacetamide.
References 205
To ascertain the mechanism, the presence of the peroxy flavin

intermediate should be identified.
Problems
1. In the reaction scheme of Eqs. 10.4 and 10.5, write down the
molecular structures of the products P1 and P2 when Phe is
used as substrate.
2. Using a steady state assumption, derive Eq. 10.6.
3. In Section 10.3.4.1, using the difference in the zero point
energy (4.8 kJ/mol) between the C–H and C–D bonds, the
primary KIE is calculated to be ~7. Confirm this calculation.
4. Section 10.3.4.1 describes that the kinetic isotope effect
(kH/kD) is equal to the pre-exponential factor ratio (AH/AD)
under the region IV. Prove this.
References
1. Koyama, H. (1982). Purification and characterization of a novel
l-phenylalanine oxidase (deaminating and decarboxylating) from
Pseudomonas sp. P-501. J. Biochem., 92, 1235–1240.
2. Koyama, H. (1983). Further characterization of a novel l-phenylalanine
oxidase (deaminating and decarboxylating) from Pseudomonas sp.
P-501. J. Biochem., 93, 1313–1319.
3. Koyama, H. (1984). Oxidation and oxygenation of l-amino acids cata-
lyzed by a l-phenylalanine oxidase (deaminating and decarboxylating)
from Pseudomonas sp. P-501. J. Biochem., 96, 421–427.
4. Mukouyama, E. B., Suzuki, H., and Koyama, H. (1994). New subunit
in l-phenylalanine oxidase from Pseudomonas sp. P-501. Arch.
Biochem. Biophys., 308, 400–406.
5. Koyama, H., and Suzuki, H. (1986). Spectral and Kinetic studies
on Pseudomonas l-phenylalanine oxidase (deaminating and
decarboxylating). J. Biochem., 100, 859–866.
6. Suzuki, H., Koyama, H., Nishina, Y., Sato, K., and Shiga, K. (1991). A
resonance Raman study on a reaction intermediate of Pseudomonas l-
phenylalanine oxidase (deaminating and decarboxylating). J. Biochem.,
110, 169–172.
7. Ohta, Y., Mukouyama, E. B., and Suzuki, H. (2006). Kinetic isotope
effect of the l-phenylalanine oxidase from Pseudomonas sp. P-501.
J. Biochem., 139, 551–555.
206 A Case Study
8. Cha, Y., Murray, C. J., and Klinman, J. P. (1989). Hydrogen tunneling in

enzyme reactions. Science, 243, 1325–1330.
9. Scrutton, N. S., Basran, J., and Sutcliffe, M. J. (1999). New insights
into enzyme catalysis. Ground state tunneling driven by protein
dynamics. Eur. J. Biochem., 264, 666–671 (review).
10. Liang, Z.-X., and Klinman, J. P. (2004). Structural bases of hydrogen
tunneling in enzymes: progress and puzzles. Curr. Opin. Struc. Biol., 14,
648–655 (review).
11. Basran, J., Sutcliffe, M. J., and Scrutton, N. S. (1999). Enzymatic H-
transfer requires vibration-driven extreme tunneling. Biochemistry,
38, 3218–3222.
12. Park, H., Girdaukas, G. G., and Northrop, D. B. (2006). Effect of pressure
on a heavy-atom isotope effect of yeast alcohol dehydrogenase.
J. Am. Chem. Soc., 2006, 128, 1868–1872.
13. Saunders, W. H. Jr. (1985). Calculations of isotope effects in elimination
reactions. New experimental criteria for tunneling in slow proton
transfers. J. Am. Chem. Soc., 107, 164–169.
14. Jonsson, T., Glickman, M. H., Sun, S., and Klinmann, J. P. (1996).
Experimental evidence for extensive tunneling of hydrogen in the
lipoxygenase reaction: Implications for enzyme catalysis. J. Am. Chem.
Soc., 118, 10319–10320.
15. Suzuki, H., Higashi, Y., Asano, M., Suguro, M., Kigawa, M., Maeda, M.,
Katayama, S., Mukouyama, E. B., and Uchiyama, K. (2004). Sequencing
and expression of the l-phenylalanine oxidase gene from Pseudomonas
sp. P-501. Proteolytic activation of the proenzyme. J. Biochem., 136,
617–627
16. Ida, K., Kurabayashi, M., Suguro, M., Hiruma, Y., Hikima, T., Yamomoto,
M., and Suzuki, H. (2008). Structural basis of proteolytic activation
of l-phenylalanine oxidase from Pseudomonas sp. P-501. J. Biol. Chem.,
283, 16584–16590.
17. Ida K., Suguro, M., and Suzuki, H. (2011). High resolution X-ray
crystal structures of l-phenylalanine oxidase (deaminating and
decarboxylating) from Pseudomonas sp. P-501. Structures of the
enzyme-ligand complex and catalytic mechanism. J. Biochem., 150,
659–669.
18. Fitzpatrick, P. F. (2010). Oxidation of amines by flavoproteins. Arch.
Biochem. Biophys., 493, 13–25 (review).
Appendix
1. Derivation of the Rate Equation by

King–Altman’s Method
The derivation of the rate equation for the Michaelis–Menten
mechanism is not hard. However, to derive the rate equation for
the complicated enzyme-catalyzed reaction, including various
enzyme species, is not easy. King and Altman introduced a simple
schematic method applicable for various enzyme-catalyzed
reactions [1–3]. For simplicity, apply the method to a following
simple mechanism:
k k k
+1
E + S 
 +2
 ES 
 +3
 EP 
  E+P (A.1)
k  k
–1

–2
k  –3
Draw the polygon having one enzyme species on each top (basic
pattern). Therefore, at least three enzyme species are required for
this mechanism.
%DVLF3DWWHUQ &DOFXODWLRQ3DWWHUQ
(6
(6 (6 (6
N>6@ N N
N
N>3@
( (3 ( (3 ( (3 ( (3
N

(1) Write an arrow on each side and the rate constant for the
respective reaction near the arrow. When a ligand is involved
in the reaction, write the rate constant multiplied with the
concentration of the ligand.
(2) Write all possible polygons without one side as above
(calculation pattern).
208 Appendix
On the basis of the patterns, determine[ES]/ [E]/e0, =[ES]/

(k+1ke–20,[S]
=and
(k++1kk–2–2k[S]
–3[P]
+ k+–2kk+1–3k[P]
+3[S])/
+ k+1Sk
[EP]/
[ES]/e0. =Here,
[ES]/
(k+1k–2
e0[S]
is
= (+the
kk+1–2kk–2
total
–3[S]
[P]+concentration
+kk k k [P]
–2+1–3+3 [S])/
+ kS kof
+1 +3 [S])/
the Senzyme species
involved. For example, [E] can be determined by multiplying the
rate constants going toward the enzyme species “E” for each
calculation pattern:
[E]
== ( + + )/e0 = (k–1k+3 + k–1k–2 + k+2k+3 )/S (A.2)
e0 .
Similarly,
[ES]/e0 = (k+1k–2[S] + k–2k–3[P] + k+1k+3[S])/S (A.3)
[EP]/e0 = (k+1k+2[S] + k+2k–3[P] + k–1k–3[P])/S (A.4)
where S is equal to the sum of the numerators of Eqs. A.2 to A.4.
S = (k–1k+3 + k–1k–2 + k+2k+3 ) + (k+1k–2[S] + k–2k–3[P] + k+1k+3[S])

+ (k+1k+2[S] + k+2k–3[P] + k–1k–3[P])

Then the rate of reaction, v, is
v/e0 = (k+3[EP] – k–3[E][P])/S

When we measure the initial rate, [P] is zero, then
v/e0 = k+3[EP]/S = k+1k+2k+3[S]/{(k–1k+3 + k–1k–2 + k+2k+3 )

+ (k+1k–2[S] + k+1k+3[S] + k+1k+2[S])}
v = k+1k+2k+3[S]e0/{(k–1k+3 + k–1k–2 + k+2k+3 ) + k+1 (k–2 + k+2k+3 )[S]}

References
1. King, E. L., and Altman, C. (1956) A schematic method of deriving

the rate laws for enzyme-catalyzed reactions. J. Phys. Chem., 60,
1375–1378.
2. Hashimoto, T. (1971) Enzyme Kinetics: Basics and Practices, Kyoritu
Publisher, Japan (in Japanese).
3. Nakamura, T. (1993) Enzyme Kinetics, Japan Scientific Societies Press
(in Japanese).
Appendix 209
2. Physical Constants

Quality Symbol SI*
Planck’s constant h 6.6261 × 10–34 J s
Avogadro constant NA 6.0221 × 1023 mol–1
Boltzmann constant k or kB 1.3807 × 10–23 J K–1
Gas constant R 8.314 5 J K –1 mol–1
Faraday constant F 9.6485 × 104 C mol–1
*The International System of Units.
3. Conversion of Units

Quality From To
Length 1Å 1 × 10–10 m
1Å 0.1 nm
Pressure 1 atm 1.01325 × 105
Pa (kg m–1 s–2)
Energy 1 cal 4.184 J
1 eV 1.6022 × 10–19 J
1 N m 1J
1J 1 kg m2 s–2
4. Prefix of Numbers and Alphabets in Greek

Number name Greek name Greek name
1 mono a alpha n nu
2 di b beta x xi
3 tri g gamma o omicron
4 tetra d delta p pi
5 penta e epsilon r rho
6 hexa z zeta s sigma
7 hepta h eta t tau
8 octa q theta u upsilon
9 nona i iota f phi
10 deca k kappa x chi
103 kilo l lambda y psi
106 mega m mu w omega
Alphabet: Upper-case is not shown.
210 Appendix
5. Useful Software and Data Banks

Jmol: an open-source Java viewer for chemical structures in 3D;
http://jmol.sourceforge.net/
PyMOL: a software tool for the analysis and visualization of protein
in 3D; http://www.pymol.org/
Caver: software tool for the analysis and visualization of tunnels and
channels in protein structures; http://www.caver.cz/
Chimera: software for the visualization and analysis of molecular
structures; good for matching two protein structures; http://www.
cgl. ucsf.edu/chimera/
Protein data bank: http://www.pdbj.org/; http://www.ebi.ac.uk/
msd/
DNA data bank Japan (DDBJ): http://www.ddbj.nig.ac.jp/index-
j.html
6. Genetic Code

Second position
First position Third position
(5-end) U/T C A G (3-end)
U/T Phe Ser Tyr Cys U/T
Phe Ser Tyr Cys C
Leu Ser Stop Stopa A
Leu Ser Stopb Trp G
C Leu Pro His Arg U/T
Leu Pro His Arg C
Leu Pro Gln Arg A
Leu Pro Gln Arg G
A Ile Thr Asn Ser U/T
Ile Thr Asn Ser C
Ile Thr Lys Arg A
Met Thr Lys Arg G
G Val Ala Asp Gly U/T
Vla Ala Asp Gly C
Val Ala Glu Gly A
Val Ala Glu Gly G
In some proteins, stop codons code amino acids: aU/TGA codes selenocystein, and
bU/TAG pyrolysine. See Chapter 6.
Solutions
Answers have been given for important and hard problems. Readers
are strongly recommended to solve problems independently and
then read the answer.
Chapter 2
1. Figure 2.2 is a plot v/V vs. log s. Therefore, Eq. 2.16 must be
changed to a function of log s:
10x
v/V =
10x + K s
where x = log s. Solve the following differential equation at

v/V = 0.5:
d(v/V ) d  10x 
=  x 
dx dx 10 + K s 
The final equation is
d(v/V ) 2.303Ks s
=
d(log s ) ( s + K s )2
At v/V = 0.5, s = Ks, we can obtain the slope, 2.303/4.

3. The lag period is the time t when p = 0 in Eq. 2.26. Thus,
t = 1/k.
4. Apply Eq. (2.23) 98.2%.
Chapter 3
2. Apply the steady state for [ES1-EP1], [F], and [FS2-FP2],
respectively, and derive the rate (v) of the enzymatic reaction
using
212 Solutions
e0 = [E] + [ES1-EP1] + [F] + [FS2-FP2]:

V k k k+4 (k–1 + k+2)
= +2 +4 ; K 1 = K mS1 =
e0 k+2 + k+4 k+1 (k+2 + k+4)

k+2(k–3 + k+4 )
K 2 = K mS2 =
k+3(k+2 + k+4 )

Chapter 4
1. Differentiation of Eq. 4.7 leads to
dV –V  H 2 – K 1K 2 
=  
dH (1+ H K1 + K 2 H )2 H 2K 1 
At the optimum pH (the proton concentration, Hop),

dV/dH = 0
Thus, H2o p = K1K2 (Eq. 4.12).
The rate Vop at Hop is determined to be given by replacing H
into Hop in Eq. 4.7:
V V
Vop = =
1+ K 1K 2
K1 + K2
K 1K 2  1+2 K2 

 K1 
 
Then
V 1+2 K 2 K1
=
Vop (1+ H K1 + K 2 H )

From this equation, H1 and H2 are calculated by introducing
V/Vop = 1/2; then
H 2 –(4 K 1K 2 + K 1 )H + K 1K 2 = 0
Assuming that a solution to the equation is H1 and H2,
(H – H1)(H – H2) = 0
Then,
H1 + H2 = 4 K 1K 2 + K 1 = K 1 + 4Hop
Solutions 213
2. From Eq. 4.27, assuming T = 298 K, the free energy of

activation can be determined by introducing 193 s–1 into kf,
then the activation entropy is calculated.
3. From Eq. 4.37 and the slope in Fig. 4.10,
d(2.303log k )
DV  = – RT
dp
d(log k )
= –2.303RT
dp
= –2.3030.0831103(ml bar K –1mol–1 )298(K)0.415× 10–3(bar–1 )
= –23.7ml
Chapter 5
lt – l∞ = (l0 – l∞) exp (–kt) (SM5.1)
l t – l∞ = (l0 – l∞) exp {–k(t + D)} (SM5.2)
Eq. SM5.1 – Eq. SM5.2,
lt – lt = (λ0 – λ∞) exp (–kt){1 – exp(–kD)}
ln (lt – lt ) = –kt + ln [(l0 – l∞){1 – exp(–kD)}]
Chapter 6
1. Let’s start with simple examples. One disulfide bond is
possible for two SH groups. For four groups, C(4,2)/2! (i.e.,
3) combinations of disulfide bond location is possible, where
C(a,b) = a!/(a–b)!b!. For six groups, C(6,2) x C(4,2)/3! (i.e.,
15) combinations. Thus, for eight SH groups, C(8,2) x C(6,2) x
C(4,2)/4! (i.e., 105) combinations. In the RNAase A, the native
enzyme has only one in 105 combinations. Consider how cells
select one combination?
2. The indole ring of Trp residue is stimulated by absorbing a
light energy to the activated state. Then the activated indole
releases an energy to return the initial low energy state.
Usually the energy is released as fluorescence. By binding with
214 Solutions
pyruvate, Trp residue on the loop moves to bind with amino

acid residues; thus, the energy on the indole is transferred
to the residues. Therefore, the fluorescence is decreased.
Chapter 7
2. The interconversion between isomers of organic compounds

is called tautomerization. Each isomer is called tautomer. The
keto-enol tautomerization may be representative:
A proton migrates in the reaction. In the amino-imino

tautomerization of TPP, N1¢ and N4¢ are involved in
tautomerization: A relay of a proton between N1¢ and N4¢
through the pyrimidine ring interconverts between the amino
and imino tautomers. In the TPP-dependent enzymes, the
imino tautomer, 1¢,4¢-iminopyrimidine, which deprotonates
the C2-H, thus, forming C2 carbanion as described in the text.
See ref. 7 in Chapter 7 for further information.
Chapter 8
2. Magnesium ions are treated as substrate and product. Mg1,
Mg2, and Mg1,2 represent that Mg ions are binding at the
Mg1, Mg2 sites, and both sites, respectively. S and P represent
the substrate peptide and the phosphorylated product,
respectively. C represents the catalytic subunit of PKA.
Solutions 215
3,4. From the assumptions,
es (e0 – x – y )s k–1
= = = K (SM8.1)
x x k+1
Then,
s
x= (e – y ) (SM8.2)
K +s 0
From the scheme given,
dy
= k+2 x – k+3 y (SM8.3)
dt
Substituting Eq. SM8.2 into Eq. SM8.3,
dy  s  k se  k s 
= k+2  (e – y ) – k+3 y = +2 0 – +2 + k+3 y (SM8.4)
dt K + s 0  K + s K + s 
Integration of Eq. SM8.4 from time 0 to t gives
k+2 se0
y= (1– e – lt ) (SM8.5)
l(K + s )
where,
k+2 s
l = k+3 +
(K + s )
By applying Eq. SM8.2, the rate of formation of P1 is
dp1 s
= k+2 x = k+2 (e – y ) (SM8.6)
dt K +s 0
Substituting Eq. SM8.5 into Eq. SM8.6, and the resulting
equation is integrated from time 0 to t,
216 Solutions
2
k+2k+3 s k2 e  s 
p1 = e0t + +22 0  (1– e – lt )
k kl K s+ s 2l  K + s 2
k e  s  – lt
p1 = +2 +3 e t + +22 0  (1– e )
l K +s 0 l K + s
 2
2 2
k k e s/(k+2 + k+3 )  k+2  s  e (1– e – lt )
 +2 +3 0 t + 2 
k+3kK + k ) 
k+2ks+3+e0 s/(  k+2k+ k+3   k+3Ks  0
+2 +3 +2  + s  e (1– e – lt )
 (k+2k +Kk+3 ) t + k + k   (k+2k+3+Kk+3 )   0
s+ +3  +2 +3 
+s
 (SM8.7)
(k+2 + k+3 )  (k+2 + k+3 ) 
p1 = v0t + (1– e – lt ) (SM8.8)
where
2 2
k+2k+3e0 s/(k+2 + k+3 )  k+2  s  (SM8.9)
v0 = ;  =   e0
s + Km  k+2 + k+3  K m + s 
k+3K .
Here, K m =
(k+2 + k+3 )
In the second equation of Eq. SM8.9, the reciprocal of square
root of both side,
1  k+3  K m  1
=1+ 1+ 
  k+2  s  e0
under the condition, k+2 » k+3

1 1  Km 
= 1+ 
 e0  s 
Thus, the crossing point of the ordinate gives the active

concentration of enzyme as shown in Box 8.2. The above
derivations are described in papers and books such as [1–4].
Probably, the derivation in [2] may be easy to understand.
References
1. Gutfreund, H., and Sturtevant, J. M. (1956) The mechanism of the

reaction of chymotrypsin with p-nitrophenyl acetate. Biochem. J., 63,
656–661.
2. Bender, M. L., Kézdy, F. J., and Wedler, F. C. (1967) α-Chymotrypsin:
Enzyme concentration and kinetics. J. Chem. Educ. 44, 84–88.
Solutions 217
3. Hiromi, K. (1978) Practical Analysis of Enzyme Reaction. 295–296.

Edited by Kodansha Scientific (Kodansha ltd, Japan) (in Japanese).
4. Nakamura, T. (1993) Enzyme Kinetics. 151–152. (Japan Scientific
Societies Press) (in Japanese).
Chapter 9
1. The DEAE group is positively charged at pH 7.0. Therefore,
the negatively charged group is able to bind with the DEAE
group. The binding strength is dependent on the total charge
of proteins.
Proteins having lower pI than the pH of the buffer; thus,
bovine serum albumin and human hemoglobin have
negative charges at pH 7.0 and bind with the cellulose. The
binding is stronger with bovine serum albumin than human
hemoglobin. On the other hand, cytochrome c is positively
charged, then does not bind with the cellulose. Order is
cytochrome c, human hemoglobin, and bovine serum
albumin.
2. NaCl dissociates into Na+ and Cl¯. These ions neutralize the
charged portions of proteins; thus, interactions with the DEAE
cellulose decrease to dissociate proteins from the cellulose.
Chapter 10
2. The steady state concentrations of the enzyme species, Eox-S,
Ered-Im, Ered-Im-O2 are constant, and the total concentration
of enzyme is
e0 = [Eox ] + [Eox-S] + [Ered-Im] + [Ered-Im-O2]
and the rate of the catalytic reaction is
v = d[P]/dt = k4[[Ered-Im-O2]
From these, we will derive Eq. 10.6.
3. kH = AH exp (–EaH/RT) (SM10.1)

kD = AD exp (–EaD/RT) (SM10.2)
kH/kD = (AH/AD) exp{(–EaH + EaD)/RT} (SM10.3)
218 Solutions
As EaD – EaH = 4.8 kJ/mol and T = 298 K, kH/kD is calculated

to be 6.94, since AH/AD = 1.
4. In the region IV of Fig. 10.4, the activationless ground
state H-tunneling occurs. Using Eq. 1.3,
ln kH = –EaH/RT + ln AH (SM10.4)
ln kD = –EaD/RT + ln AD (SM10.5)
Subtracting Eq. SM10.5 from Eq. SM10.4 gives
ln kH – ln kD = (–EaH + EaD)/RT + ln AH – ln AD
As (–EaH + EaD) is zero under the conditions,

kH/kD = AH/AD.
“The enzyme is a classic but very important material that everybody in the field of biology,
medicinal chemistry, biotechnology, and medicine must be familiar with. It is the extreme
feature of protein that works in the cells. This concise book covers classic and modern
enzymology and, therefore, is an excellent guide for those who possess the basic knowledge
of chemistry and want to proceed to advanced courses.”
Prof. Takeshi Nishino
University of Tokyo, Japan
How Enzymes Work

“This carefully written book provides very useful information on enzymes and will immensely
benefit not only undergraduate and graduate students but also researchers interested in
enzymes.”
Dr. Hitoshi Nakamoto
Saitama University, Japan
For a long time, enzymes have been studied by measuring their activity, which has
led to the advancement of “enzyme kinetics.” In recent years, the mechanism of
enzyme reaction has been explained in detail on the basis of the 3D structure. Genetic
engineering and the 3D structural analysis of enzymes contribute to these advancements
in enzymology. This book starts with an introduction to various enzymes to show how
interesting enzymes are, which is followed by historical kinetic studies on enzymes and
the overall and rapid-reaction kinetics. The subsequent topics describe the basics of
protein structure, the control of enzyme activity, and the purification of enzymes. A case
on the kinetic and structural studies of l-phenylalanine oxidase is also presented. There
are many good books on enzyme kinetics, but few describe their kinetic and structural
aspects. This book deals with both and contains many references that can be good
sources for further reading. It is handy and is especially helpful for beginners. A number
of figures, including some with stereo expression, facilitate observing the 3D structure
of enzymes.
Haruo Suzuki is professor emeritus at Kitasato University, Tokyo, Japan,

a councilor of the Japanese Biochemical Society, and a member of
the Japan Society for Bioscience Biotechnology and Agrochemistry. A
biochemist, he graduated from the Department of Chemistry, Tokyo
Metropolitan University, in 1966 and received his DSc from the Division of
Suzuki
Biophysics and Biochemistry, the Graduate School of Science, University
of Tokyo, in 1971. He worked as a postdoctoral fellow in the Department
of Pathology, University of California at San Diego, from 1971 to 1973. He worked at
the Institute for Developmental Research, Aichi Prefectural Colony, Japan, from 1973 to
1978, Kitasato University School of Medicine, Japan, from 1978 to 1994, and Kitasato
University School of Science from 1994 to 2007. Prof. Suzuki’s research interests focus on
the computer analysis (QM/MD) of enzyme catalysis.
V424
ISBN 978-981-4463-92-8

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“The enzyme is a classic but very important material that everybody in the field of biology,

Haruo Suzuki is professor emeritus at Kitasato University, Tokyo, Japan,

The Rise of Modern Wind Energy

No claim to original U.S. Government works

International Standard Book Number-13: 978-981-4463-93-5 (eBook - PDF)

2. Overall Reaction Kinetics 17

2.4 Kinetic Parameters 30

4. Effect of pH, Temperature, and High Pressure on

4.4 Effect of Pressure 66

5. Measurement of Individual Rate Constants 75

7. Active Site Structure 117

8. Control of Enzyme Activity 141

8.2.1 Activation of Enzymes by Cleavage of

9. Preparation of Enzyme 171

10. A Case Study: l-Phenylalanine Oxidase (Deaminating

10.3 Catalytic Properties of PAO 188

About three years ago, I received an e-mail from Mr. Stanford

to picosecond level. In Chapter 8, I make a brief mention of the

Foods are digested in the human body to glucose, fatty acids,

1.1 General Properties of Enzyme

How Enzymes Work: From Structure to Function

yeast in Greek. Many enzymes have name with suffix—ase, which

1.1.1 Enzyme Specificity

1.1.2 Rate Enhancement

energy barrier (Fig. 1.1). The energy is called activationdenergy,

Figure 1.1 Energy profiles of uncatalyzed and catalyzed reactions.

Table 1.1 Rate enhancement by enzymes

Enzymes Rate enhancement

Determination of the activation energy: Integrating Eq. 1.1,

where A is pre-exponential factor. Logarithm of Eq. 1.2 results in

1.2 Examples of Enzyme

the neuronal transmission. In this transmission, the signal from one

Figure 1.3 Neurotransmission Action potential reaches prosynaptic

Thus, the signal transmits presynaptic to postsynaptic cells.

CH3COOCH2CH2N+(CH3)3 + H2O  CH3COO– + H+ + HOCH2CH2N+ (CH3)3

To perform the rapid transmission of signal, the esterase has

1.2.2 Gastric Juice and Proton Pump

CO2 + H2O  H+ + HC​O​–3 ​​ (1.4)

The catalytic activity of carbonic anhydrase is one of the

The proton pump, H+, K+-ATPase, carries H+ through the

membrane. The reaction mechanism of H+, K+-ATPase is shown

1.2.3 Genetic Test of Alcohol Sensitivity and DNA

Ingested alcohol (ethyl alcohol) is oxidized by alcohol dehydrogenase

C2H5OH  CH3CHO  CH3COO– (1.5)

Humans have more than 12 aldehyde dehydrogenase genes

Glu (GAA)  Lys (AAA) (1.6)

Therefore, if we perform 20 cycles of PCR, we will obtain 106-fold

Figure 1.6 Amplification of dsDNA by polymerase chain reaction.

In the case of aldehyde dehydrogenase (ALDH2), the exon 12 of

Figure 1.7 Agarose gel electrophoresis pattern of the PCR-amplified

1.2.4 Enzyme Sensor Determination of Glucose

Glucose + O2  Glucono-δ-lactone + H2O2. (1.7)

H2O2 produced oxidizes the colorless chromogen to the colored

Chromogen + H2O2  Chromophore + H2O (1.8)

Familiar chromogens are ABTS {2,2¢-azino-bis(3-

by Clark [16]. The sensor is composed of the Clark-type oxygen

O2 + 4H+ + 4 e–  2H2O (1.9)

At anode (silver), solid silver is oxidized:

4Ag + 4Cl–  4AgCl + 4e– (1.10)

By this electrode, one can easily determine the glucose content as

Figure 1.8 Electrode for glucose sensor. GO, polyethylene membrane

Overall Reaction Kinetics

CO2 + H2O  H+ + HCO–3 (1.4)