Concise Biochemistry: Fundamental Principles: March 2016

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concise
BIOCHEMISTRY Fundamental Principles
Aditya Arya, PhD
Second Edition
Concise Biochemistry
Fundament al P rinciples
Fundament al P rinciples
2 nd E dition
Digitally signed by Dr. Aditya Arya
Dr. Aditya DN: cn=Dr. Aditya Arya, c=IN,

o=Drawing Pin Publishing, ou=DPP,
email=drawingpinpublishing@gmail.
com
Arya Reason: Dr Arya is Original Author of

this book.
Date: 2018.01.23 15:37:33 +05'30'
Aditya Arya, PhD

Defence Research and Development Organization
New Delhi, India
Life Sciences
New Delhi, India
Life Sciences
CONCISE BIOCHEMISTRY FUNDAMENTAL PRINCIPLES
Second Edition
Paperback (380 pages)
ISBN 000-0-0000-0000-0
Copyright © 2017. All Rights Reserved. Drawing Pin Publishing.
All rights reserved. No part of this publication may be reproduced or transmitted

in any form or by any means, electronic or mechanical, including photocopying,
recording, or any other storage and retrieval system, without prior written
permission of the publisher. All the copyright related queries may be directly sent
to the publisher at drawingpinpublishing@gmail.com
Disclaimer
Although, utmost care has been taken to avoid any errors in the preparation of this
book. Authentic and original resources have been referred, however in case of any
discrepancies or loss of any kind due to incorrect content authors or publishers
shall not be responsible. Also, the concepts of biochemistry mentioned in the book
are not for clinical advice or suggestions.
About cover image: A part of structure of human serum albumin protein, originally
submitted to RCSB by Sugio, S. et al.
Cover Design: Dr. Aditya Arya

Typesetting Editor: Rakesh Bamrara
Production Manager: Ganga Ram Arya
Copyediting Editor: Anamika Gangwar
Post production manager: Shashank Arya
Printed in India
To Anjali
Preface
I am delighted to present this book entitled “Concise Biochemistry: Fundamental Principles – 2e”especially prepared for
the preparation of various nationwide exams for higher education and research as well as university coursework. The
second edition was quickly revised after huge success of 1st edition, few more questions and context was included
in this edition and very soon 3rd revised edition will come with all illustration re-designed and some current topics
included. The book will cater as a concise teaching guide, learning aid for university exams and a general Biochemistry
textbook as well. The students may opt for a number of Biochemistry books for the study material for CSIR-NET, GATE,
IIT-JAM, etc. written with the objective of providing a support to clear these exams but the books available in the
market for the above said purpose only focus on MCQs and lack the conceptual knowledge. However, I should not
include my critical evaluation on few of the finest books on biochemistry including Lehninzer Biochemistry by Nelson
and Cox, Biochemistry by Voet and Voet Harpers Biochemistry and Lippincott’s illustrated reviews, which have always
been a source of inspiration and guideline for me to write this book and I must do acknowledge these authors for using
their ideas and analogies at some instances in this book. I would also recommend the students to read these books for
any further elaboration on the subject. As an additional advantage, this book is concise and focused for competitive
exams distinguishing from other books. This book has been divided into three parts, the first part is essential chemistry
for biology that includes atomic structure, intermolecular interactions, bioenergetics and kinetics. These topics are
generally omitted or curtailed by many biochemistry books but these are very important and frequently asked in exams.
The students often find these topics difficult and search of quality content proves to be a mere wastage of time. Second
part of the book contains description on all the key biomolecules, some new information has also been added on all the
biomolecules especially based on the trend of previous exams. The third part consist of metabolism and its regulation by
enzymes. Although the topic metabolism has been curtailed, yet, I have included some important points in metabolism
like tracing molecules and organ specific metabolism which will provide additional benefit to the students. In order to
customize this book for the preparation of the competitive exams, after each chapter one page concept map, solutions
to previous year questions from that topic and some of the high yielding facts have been added. The purpose of these
sections is to provide a memory aid to the students. Additionally an exam index and statistics of exam questions from
various papers have also been provided in the beginning of each chapter. I presume this book will render a great support
and proved to be the best study material towards the preparation for aforesaid competitive examinations. Initially I would
extend my sincere acknowledgment to my Biochemistry teachers Dr. CKS Shrotri (Boston College) and Prof. GS Selvam
(Madurai Kamaraj University), who introduced me this subject at undergraduate and postgraduate level respectively.
I am extremely thankful to Dr. Mohit Gupta and Mr. Rahul Raj for his constant motivation and whole hearted support
for the development of this book and supporting its publication. I also acknowledge the efforts and inputs made by my
colleagues Anamika Gangwar, Shikha Jain, Subhojit Paul, Amit Kumar and Nassruddin and many other students who
have contributed their ideas. I am grateful to Dr. Atul Verma for English editing. My Grandfather, parents and younger
brother Shashank have immense contribution in publishing this book which include their emotional and motivational
support. I am also indebted for the cooperation andsupport which was rendered unconditionally including technical
assistance by Mr. Ashutosh, Mrs. Shruti Jain, Ms. Heena Baluja and Mr. Suraj. I look forward for kind suggestions from
the valuable readers and students for further improvement in content and layout.
With best Wishes.

—Dr. Aditya Arya
Contents
Section 1: Essential Chemistry in Biology
Chapter 1: Atomic Structure and Chemical Bonding��3–22

1.1 Introduction .............................................................................................................................................. 3
1.2 Chemical bond ......................................................................................................................................... 8
chapter 2: Mole Concept and Concentration Terms��23–36

2.1 Introduction ............................................................................................................................................ 23
2.2 Definition of mole .................................................................................................................................. 23
2.3 Common concentration terms: How to make a choice?........................................................................ 25
2.4 Dilution of solutions .............................................................................................................................. 31
chapter 3: Concept of pH and Biological Buffers��37–59

3.1 Introduction ............................................................................................................................................ 37
3.2 Concept of equilibrium........................................................................................................................... 37
3.3 Concept of pH ........................................................................................................................................ 42
3.4 Buffers and buffering capacity.............................................................................................................. 44
3.5 Major biological buffers ........................................................................................................................ 48
Chapter 4: Bioenergetics and Energy Coupling�� 60–82

4.1 Introduction............................................................................................................................................. 60
4.2 Laws of thermodynamics: A contrast between chemistry and biology............................................... 61
4.3 Some examples of Gibbs law in biological systems............................................................................. 69
4.4 Major energy transducers ..................................................................................................................... 73
Chapter 5: Chemical Kinetics & Colligative Properties��83–92

5.1 Introduction ............................................................................................................................................ 83
5.2 Mathematical Expression for velocity of a reaction............................................................................. 84
5.3 Colligative Properties ............................................................................................................................ 87
Section 2: Biomolecules: Structure and Function
Chapter 6: Amino acids: Structure and Properties�� 95–112

6.1 Introduction............................................................................................................................................. 95
6.2 Basic structure of amino acids and associated nomenclature............................................................. 96
6.3 Classification of amino acids................................................................................................................. 98
6.4 Detailed description of standard amino acids..................................................................................... 100
6.5 Titration curves of amino acids and pI ............................................................................................... 104
Chapter 7: Proteins and their conformations��113–133

7.1 Introduction............................................................................................................................................113
7.2 Peptide bond formation and torsional rotation.....................................................................................115
7.3 The Ramachandran plot.........................................................................................................................117
7.4 Protein conformations and levels of folding.........................................................................................118
Chapter 8: Proteins: Classification & model examples�� 134–146

8.1 Introduction........................................................................................................................................... 134
8.2 Structural classification of proteins .................................................................................................... 134
8.3 Keratin ................................................................................................................................................. 135
8.4 Silk fibroin (beta keratin)...................................................................................................................... 137
8.5 Collagen................................................................................................................................................ 138
8.6 Ribonuclease ....................................................................................................................................... 140
8.7 Hemoglobin & Myoglobin .....................................................................................................................141
8.8 Public repositories for proteins structure and sequences................................................................... 143
Chapter 9: Carbohydrates: Structure and Functions��147–172

9.1 Introduction........................................................................................................................................... 147
9.2 Basic physical properties of carbohydrate structure ......................................................................... 148
9.3 Structural diversity in carbohydrates. ................................................................................................ 152
9.4 Glycoconjugates................................................................................................................................... 166
Chapter 10: Lipids: Structure and Biological Functions�� 173–194

10.1 Introduction ........................................................................................................................................ 173
10.2 Fatty acids are building blocks of several lipids .............................................................................. 173
10.3 Simple lipids........................................................................................................................................ 179
10.4 Complex lipids..................................................................................................................................... 183
10.5 Derived lipids...................................................................................................................................... 187
Chapter 11: Nucleic Acids: Structural Biochemistry�� 195–218

11.1 Introduction ........................................................................................................................................ 195
11.2 Components of nucleic acid .............................................................................................................. 196
11.3 DNA double helical structure............................................................................................................. 201
11.4 Topology of DNA................................................................................................................................. 207
11.5 RNA: Types and secondary structural motifs ....................................................................................211
Chapter 12: Stabilizing-interactions in Biomolecules��219–236

12.1 Introduction .........................................................................................................................................219
12.2 Specific linkages in biological systems..............................................................................................219
12.3 Stabilizing interactions in proteins ................................................................................................... 221
12.4 Denaturation and renaturation kinetics of proteins ......................................................................... 223
12.5 Stabilizing interactions in DNA.......................................................................................................... 226
12.6 Denaturation and renaturation kinetics of DNA................................................................................ 228
x
Table of Contents
Section 3: Metabolism and its Regulation
Chapter 13: Global View of Metabolism��239–253

13.1 Introduction......................................................................................................................................... 239
13.2 Energy and thermodynamic considerations .......................................................................................241
13.3 Metabolic pathways are integrated, not discrete............................................................................. 242
13.4 Metabolic pathways are anatomically heterogeneous .................................................................... 243
13.5 Global regulation of metabolism........................................................................................................ 245
13.6 How to study metabolism ................................................................................................................. 247
Chapter 14: Metabolism of Biomolecules��254–295

14.1 Metabolism of carbohydrates ........................................................................................................... 254
14.2 Metabolism of Lipids.......................................................................................................................... 266
14.3 Metabolism of proteins and amino acids.......................................................................................... 276
14.4 Metabolism of nucleotides ................................................................................................................ 287
Chapter 15: Enzymes I: Principles of Catalysis��296–316

15.1 Introduction ........................................................................................................................................ 296
15.2 Timeline of enzymology research...................................................................................................... 296
15.3 Components of an enzymes............................................................................................................... 298
15.4 Classification of enzymes................................................................................................................... 299
15.5 Principles of enzyme catalysis........................................................................................................... 301
15.6 Types of enzyme catalysis ................................................................................................................ 304
15.7 Factors affecting enzyme activity ..................................................................................................... 306
15.8 Vitamins: Cofactors of enzymes........................................................................................................ 307
chapter 16: Enzymes II: Kinetics��317–333

16.1 Introduction ........................................................................................................................................ 317
16.2 Michaelis-Menten Kinetics.................................................................................................................318
16.3 Isozymes ............................................................................................................................................ 322
16.4 Measures of enzyme efficiency ........................................................................................................ 325
16.5 Kinetics of bisubstrate reactions....................................................................................................... 327
16.6 Pre-Steady state kinetics................................................................................................................... 328
Chapter 17: Enzymes III: Regulation�� 334–352

17.1 Introduction ........................................................................................................................................ 334
17.2 Overview of Enzyme regulation......................................................................................................... 335
17.3 Allosteric regulation............................................................................................................................ 335
17.4 Enzyme regulation by covalent modification .................................................................................... 340
17.5 Enzyme regulation by limited proteolysis cleavage .......................................................................... 343
17.6 Enzyme regulation by selective inhibitors.......................................................................................... 344
xi
Appendix 1: List of Common Biochemical Tests�� 353–356
Appendix 2: List of Common Inhibitors��357–360
Appendix 3: Reference values in Blood Tests�� 361
appendix 4: Credits and Suggested Readings��362–366
xii
chapter 3: Concept of pH and Biological Buffers 47
The above expressions are used to calculate pH of a buffer when the

concentration of salt and acid are known. Now, someone wants to prepare trick to Remember
a buffer from acetic acid and sodium acetate (pKa of acetic acid is 4.76), any
desired pH could be obtained by modulating ratio of salt and acid. Varying S comes later in alphabet, so it is senior
TRICK TO REMEMBER
the ratio as 1, 10, 100, 1000 we may get pH of buffer as 4.76, 5.76, 6.76 and and sits above, acids sits below, reversing
7.76. Also, by changing the ratio to 0.1, 0.01, 0.001 we may get pH as 3.76, the order becomes a point of error in many
questions during exam.
2.76 and 1.76 respectively.
Based on this fact we can make a buffer of any pH just by changing the ratio of salt and acid and it is true than we can work with
Further during the entire text some tricks to remember

single type of buffer in all the experiments. But that does not mean a buffer will be equally effective and efficient to resist the
change in pH in all the conditions. Let us understand the conditions when a buffer will work optimally or show its best potential.
the difficult topics have also been added, which

3.4.4 conditions for best buffering capacity
Recall the principle of buffering as shown in the Figure 3.5. We observed that presence of salt is preventing the reaction from
reverting back and thus eqimolar quantities of salt and acid will be optimal to have best buffering capacity on either direction
(pH could be resisted with equal magnitude on both acidic and basic sides) in such condition when ratio of salt and acid is one.
Log Salt/Acid will be 0 (Log 1 5 0).

is unique feature of this book usually not found in
So keeping this in HH equation we get pH 5 pKa
Therefore, a buffer will show its best buffering capacity when pH of the buffer is equal to pKa of the acid used in the buffer.
standard texts.
So, as discussed in previous section, despite the fact that we can make a buffer of any desired pH from various combination of
acetic acid and sodium acetate, a buffer of pH 4.76 will have best buffering activity. For this reason, we have different type of
buffers during different type of biochemical reactions or experiments. (Acetic acid buffer for pH 4-5; tartaric acid buffer 2-4; Tris
base buffer for pH 12-14 etc.)
3.4.5 effect of dilution on buffers.

Although acids and bases lose their strength on dilution depending on how many times the solutions are being diluted.
Dilution will also dilute buffers by (if we diluted 10 ml of buffer to 90 ml of solution, buffer will be diluted by 10 times). But
interestingly all the components of the buffer will be diluted by same magnitude. So as per the HH equation if both salt and
acid are diluted by same factor the value of pH will still remain same. Therefore, pH of a buffer does not change on dilution.
Under certain conditions when the dilution is too large such that the H1 ions contributed by water are larger than the H1 ions
present in the buffer, the pH may change, in such a situation we need to add the H1 ions of water and H1 ions of buffer and
recalculate the pH. (Practically, we need to dilute buffer several million times to achieve this state!)
22 Concise Biochemistry
Some practice problems on HH equation

q: Calculate the pH of a mixture of 0.25 M acetic acid and 0.1 M Sodium acetate. The pKa of acetic acid is 4.76
HigH YiELdiNg fActS
High Yielding Facts

sol: As per the HH equation
According to Bohr and Bury, the maximum number of electrons that can be accommodated in any energy level
Salt of an atom is given by the formula 2n 2, where ‘n’ represents the number of the energy level.
pH = pKa + log
Acid
In order to exist independently by itself an atom must have eight electrons in its outermost shell two electrons
pH 5 4.76 1 Log 0.1/0.25 5 4.76 – 0.398 5 4.36 if there is only one shell. This is the octet rule.
q: Calculate the pH in the preceding problem if the mixture consist of 0.1 M acetic acid and 0.25 M sodium acetate? Atoms try to attain stable configuration (completing their outermost shell) either by losing, gaining or sharing
sol: As per the HH equation electrons.
Salt Coordinate bond is a covalent bond in which the shared pair of electrons is contributed by only one of the two
pH = pKa + log atoms.
Acid
pH 5 4.76 1 Log 0.25/0.1 5 4.76 1 0.398 5 5.16 Theories of chemical bonds go back a long time. One of the first was developed by Roman poet Lucretius
(95-55 BC), author of De Rerum Natura (title means “on the nature of things”).
Van der Waals forces were named in honor of the Dutch physicist Johannes Diderik van der Waals (1837–1923),
ocopying and distribution will be treated under Law © 2016. Aditya Arya, Grassroots Academy. All Rights reserved. Unauthorised Photocopying and distribution will be treated under Law who investigated the weak non-chemical bond forces between molecules.
The Stern–Gerlach experiment of 1922 provided further evidence of the quantum nature of the atom. When a
beam of silver atoms was passed through a specially shaped magnetic field, the beam was split based on the
direction of an atom’s angular momentum, or spin.
The electron is by far the least massive of these particles at 9.11 3 10 −31 kg, with a negative electrical charge
and a size that is too small to be measured using available techniques.
Protons have a positive charge and a mass 1,836 times that of the electron, at 1.6726 3 10 −27 kg. The number
of protons in an atom is called its atomic number.
high yielding facts

Neutrons have no electrical charge and have a free mass of 1,839 times the mass of the electron, or 1.6929 3 10−27
kg, the heaviest of the three constituent particles.
In the Standard Model of physics, electrons are truly elementary particles with no internal structure. However,
both protons and neutrons are composite particles composed of elementary particles called quarks.
The chapter ends with a section called high There are two types of quarks in an atom, each having a fractional electric charge. Protons are composed of
two up quarks (each with charge 12 ⁄3) and one down quark (with a charge of −1⁄3). Neutrons consist of one
up quark and two down quarks.
yielding facts, this includes some of the The quarks are held together by the strong interaction (or strong force), which is mediated by gluons. The
protons and neutrons, in turn, are held to each other in the nucleus by the nuclear force.
unique small points about the topic that may

be remembered by the students especially
for memory based questions asked in many
competitive examinations.
References, Suggested readings and credits for this chapter are given at the end of this book
© 2016. Aditya Arya, Grassroots Academy. All Rights reserved. Unauthorised Photocopying and distribution will be treated under Law
Chapter 1: Atomic Structure and Chemical Bonding 21
QuEStioNS froM PrEViouS ExAMS

Questions from Previous Exams
Q1: Which of the following non-covalent interaction between two non-bonded atoms A and B is most sensitive to the
distance between them? [CSir june 2012]
a. A and B are permanent dipoles and are involved in salt bridges
b. A and B are fully ionized and are involved in salt bridges
c. A and B are uncharged and repel each other
d. A and B are uncharged and attracted each other]
Solution: Van der Waals interactions at a distance below 5 Å become highly repulsive and the magnitude of
these repulsive van-der-waals interactions is related inversely to 12th power of distance between two charges
(or nuclei of atoms) therefore c is correct answer.
Q2: On the molar scale which of the following interactions in a non-polar environment provides highest contribution
question from previous exam
to the bio-molecule? [CSir net dec 2011]
a. Van der Waals interaction
c. Salt bridge
b. Hydrogen bonding
d. Hydrophobic interaction This is followed by another important segment
Solution: Hydrogen Bonds are associated with water (in hydrophillic environment), Salt bridges are also
electrostatic in nature and therefore depend upon the hydrophillic environment. Hydrophobic interaction are also
a result of repulsion from hydrophillic groups. Van der waals interactions are the only interactions that provide containing previous year questions from CSIR-
highest contribution to biomolecule.
Q3: If van der Waals interaction is described by the following relation,

A B qq
NET from that particular chapter, providing
∆ GVan = 12 - 6 + 1 2
r r r
Where DGVan is the free energy of the van der Waals interactions, A and B are constants, r is the distance
students a recap of what need to be focused
between two non-bonded atoms 1 and 2, and q1 and q2 are partial charges on the dipoles 1 and 2. In this relation,
the parameter A describes:
a. electron shell attraction b. electron shell repulsion
[CSir net dec 2011]
in the chapter and detailed solution of each
previous year question.
c. dipole-dipole attraction d. dipole-dipole repulsion
Solution: In this formula the net Gibbs free energy formula for Van der waals interaction is given, in
which the initial two components are called as Lenard Jones potential or L-J potential. This is expressed in a
simplified form as DG 5 A/r12 – B/r 6 and hence, is also known as 6–12 potential. A/r12 is predominant at short
distances and hence, represents the short-range repulsive potential due to overlap of electron orbitals and
B/r 6 is dominant at longer distance and hence, is the long range attractive potential.
Q4: In proteins, hydrogen bonds form as follows: Donor (D)-H---Acceptor (A). Hydrogen bond is more favorable if the
angle between D-H and A is [CSir-net 2014]
a. ,90° b. 180°
c. .180° d. 120°
Solution: The angle between the D-H bond and the H---A hydrogen bond should be close to 180° for a strong Additionally, as an innovative approach, some of the
hydrogen bond, hence b is correct answer.
difficult topics are aided with online support. Readers may

visit you tube channel of author or the publisher to watch
curtailed classroom videos for clarity.
ocopying and distribution will be treated under Law © 2016. Aditya Arya, Grassroots Academy. All Rights reserved. Unauthorised Photocopying and distribution will be treated under Law
xiv
Section
1
Chapter 1: Atomic Structure and Chemical Bonding 3–22
Chapter 2: Mole Concept and Concentration Terms 23–36
Chapter 3: Concept of pH and Biological Buffers 37–59
Chapter 4: Bioenergetics and Energy Coupling 60–82
Chapter 5: Chemical Kinetics & Colligative Properties 83–92
1
2
chap t er
Atomic Structure and
Chemical Bonding
1
Learning ObjectivesExam Index 


•• Atom, sub-atomic particles and their organisation.
•• Various models of atom, salient features and drawbacks.
•• Chemical bonds, their formation and types.
•• Comparison between all types of interactions in terms of their strength.
•• Distance dependence on the magnitude of interaction/bond.
1.1. Introduction
Biomolecules are characterised by their biological origin and may appear
to be more complex than basic chemical entities often seen in chemistry Interesting Fact
textbooks, yet their fundamental constituents are atoms and their chemical
properties are governed by the same rules of chemistry. The “God particle” is the nickname of a subatomic
particle called the Higgs boson. In layman’s terms,
In order to understand the stability of biomolecules, their interactions and
different subatomic particles are responsible for
the mechanism of biochemical reactions, it is essential to revisit the atomic giving matter different properties. One of the most
structure and basic atomic/molecular interactions that are essential for the mysterious and important properties is mass.
existence of every chemical entity in this universe. In this chapter, we will Some particles, like protons and neutrons, have
also focus on biological relevance of atomic structure and their interactions. mass. Others, like photons, do not. The Higgs
boson or “God particle,” is believed to be the
The word atom has originated from the Greek word ‘a-tomio‘ which means particle which gives mass to matter. The “God
uncutable or non-divisible. The details of the atom could be elucidated only particle” nickname grew out of the long, drawn-
after the discovery of sub-atomic particles such as electron (JJ Thomson), out struggles of physicists to find this elusive
proton (Goldstein) and neutrons (Chadwick). Since early 1800, the atomic piece of the cosmic puzzle. What follows is a very
structure has been extensively revised and elaborated by several classical brief and simplified explanation of how the Higgs
boson fits into modern physics, and how science
experiments. Chronologically, initial model was plum pudding model
is attempting to study it.
given by JJ Thomson, followed by Rutherford’s atomic model, which
was later refined by Neil’s Bohr. The most recent model of atom is based
on quantum mechanics and called as quantum mechanical model. Let us
now understand the key ideas proposed in these model and refine our understanding about the structure of atom.
1.1.1. Plum Pudding Model

JJ Thomson, in 1898, proposed that an atom possesses a spherical shape (radius ~ 10 –10 m) in which the positive charge is
uniformly distributed. The electrons are embedded into it in such a manner as to give the most stable electrostatic arrangement.
This may be understood similar to plums as electrons present in positively charged pudding or seeds as electrons present in
3
Chapter 2: Mole Concept and Concentration Terms
self-test
Q: How much water do you take in a day? Calculate the number of water molecules and hydrogen atoms taken by you
each day in the form of water?
[Hint: Each 18 ml is equivalent to 18 g (as density of water is 1g/ml) and therefore calculate the mass of water you
consume every day and then the number of moles by using relation explained above in text]
Now after understanding the basic concept of mole and solving some numerical problems let us try to understand the
concentration terms. A concentration term is a method of representing the relative amount of solute in a solution with respect
to the solvent. There are more than 10 different ways to represent concentration terms but how to make a choice and when to
choose a particular concentration term will be discussed in the following section.
2.3 Common concentration terms: How to make a choice?

There are several ways of representing the concentration terms, the choice of concentration term depends upon the need of the
experimenter and kind of the experimental conditions. Usually the concentration terms that are used for pharmaceutical purpose
or displayed on labels of food need to be simple and easy to understand, therefore, most of them are percentage concentration
terms. While those which are used for the laboratory purpose need to display the number of moles or particles as one need
to know these parameters for knowing the stoichiometry of the reaction. The third class of concentration terms is for very
small magnitude and especially used for environmental applications or toxicological studies, such as representing the level of
pollutants. IUPAC recommends Molarity as a standard concentration term. Figure 2.2 outlines the categories of concentration
terms on the basis of usage.
Fig. 2.2. Classification of major types of concentration terms based on their usage.
Online Support
You tube channel: video 2.2: Concentration terms
25
self-test
Q: Which is of the following is true about the reactant and product concentration at equilibrium?
a. Reactant 5 product b. Reactant>Product
c. Reactant<Product d. Any of the three
[Hint: If you were wondering that the answer is ‘a’ you may have to revisit the meaning of equilibrium].
An equilibrium does not always mean that half of the reactants have converted into products, rather it is the time-
point when no net conversion in any direction is taking place and therefore the concentration of reactants or products
is unchanged. Equilibrium can be achieved at any time depending on intrinsic nature of reactant and product and their
energetics. It may be achieved just a moment after the reaction started, or somewhere in the halfway of the reaction or in
other cases towards the completion of reaction. Now all this description could be represented by a physical parameter called
equilibrium constant.
An equilibrium constant is defined as the ratio of concentration of products and reactants at that time when no net
conversion is observed. It is represented by Keq or K (In this text, use of K is avoided as K is also used to represent rate
constant which has different meaning, however the distinction between two parameters may be made by use of lowercase
k or Greek letter (k) for rate constant and uppercase (K) for equilibrium constant).
For a chemical or physical process A 1 B C 1 D:
[C ][D ]
K eq =
[ A ][B ]
The value of Keq may be 1 (reactants equal to products), smaller than 1 (reactants more than products) or larger than 1 (products
more than reactants). Therefore we can understand the location of equilibrium on the reaction progress bar as shown in Fig. 3.3.
Equillibrium Acheived in the Equillibrium Acheived Equillibrium Acheived

begenning of the reaction in the mid of the reaction in the end of the reaction
Keq < 1 Keq = 1 Keq > 1
Progress of Reaction
Only Reactants Reactants equal Only Products

Present to Products Present
Fig. 3.3. Relationship between the value of equilibrium constant and position of equilibrium over the progress plot.
Remember, Equilibrium constant is not representing how fast the equilibrium is being attained as it would be dependent on
the kinetics or the rate of reaction. Reaction of two different velocities can have same Keq values. For determining the rate of
reaction we may need to look at the reaction kinetics. Now let us look at some of the variants of equilibrium constant that are
commonly used in biochemistry.
3.2.2. Acid Dissociation constant

An acid dissociation constant, Ka, (also known as acidity constant, or acid-ionization constant) is a quantitative measure of the
strength of an acid in solution. It is the equilibrium constant for a chemical reaction known as dissociation in the context of acid-
base reactions. The larger the Ka value, the more dissociation of the molecules in solution and thus the stronger the acid.
The equilibrium of acid dissociation can be written symbolically as:
HA  H+ + A-
40
Chapter 3: Concept of pH and Biological Buffers
Where HA is a generic acid that dissociates by splitting into A−, known as the conjugate base of the acid, and the hydrogen ion
or proton, H1, which, in the case of aqueous solutions, exists as the hydronium ion—in other words, a solvated proton (H3O1).
The chemical species HA, A− and H1 are said to be in equilibrium when their concentrations do not change with the passing
of time. The dissociation constant is usually written as a quotient/ratio of the equilibrium concentrations (in mol/L), denoted by
[HA], [A−] and [H1]:
The larger the Ka value, the more dissociation of the molecules in solution and thus, the stronger the acid.
3.2.3 Base Dissociation constant

A base dissociation constant, Kb, (also known as basicity constant, or base-ionization constant) is a quantitative measure of the
strength of a base in solution. It is the equilibrium constant for a chemical reaction in the context of dissociation of a base such
as breakdown of NaOH into Na1 and OH –.
This reaction can be written symbolically as:
BOH  B+ + OH-
Where BOH is a generic base that dissociates by splitting into B1, and OH – . B1 is known as the conjugate base of BOH. The
dissociation constant is usually written as a quotient of the equilibrium concentrations (in mol/L), denoted by [BOH], [B1] and
[OH –].
The larger the Kb value, the more dissociation of the molecules in solution and thus the stronger the base.
3.2.4. Representing multiple equilibria

In case of molecules having more than one ionisable groups (ionizable H1 ions/OH – ions), each equilibria can be represented as
K1, K 2, K3 and so on. e.g.
For each of the equilibria of phosphoric acid
H3PO4  H2PO-4 + H+  HPO24- + H+  PO34- + H+
We may represent the equilibrium constant of each step as K1, K 2 and K3 respectively. Also in case of biomolecules such as amino
acids the equilibrium for ionization of side chain is represented by KR (R means side chain).
3.2.5. p- Function for equilibrium constants

The realistic values of equilibrium constant in biological as well as chemical systems are very small (such as 10 –4) and therefore,
it is difficult to compute them in associated mathematical problems (e.g. adding 2.5310 –5 to 4.8310 –7 is much difficult than
adding 4.6 and 7.3, which are respective negative log values), so an improved parameter called pKeq has been introduced
which is defined as negative log of equilibrium constant. This conversion makes the values simpler (such as 10 –4 is converted to
4 which is much easier to compute).
pKeq5 –log Keq Similarly, pKa 5 –log Ka; pKb 5 –log Kb; pK1 5 –log K1; pK2 5 –log K2; pKR 5 –log KR
•• Thus larger the Ka value, stronger an acid and lower will be its pKa
•• Smaller the Ka value weaker will be the acid and larger will be the pKa
•• Similarly the –log value for multiple equilibria may be represented as pK1, pK 2, pKR etc.
Relationship between degree of dissociation (alpha, a) and acid dissociation constant (Ka)
Example AH  A– 1 H1 if degree of dissociation is 10% (or 10/100 5 0.1) then at equilibrium 0.1 will be product (each A1 and
H1 will be 0.1) undissociated acid will be 0.9 [note that here concentration of each of the ionised components are in terms of
solution so they will be 10% each and NOT 5%].
41
Chapter 3: Concept of pH and Biological Buffers
Alteration in Physiological pH
Pathological Conditions
Acidosis Alkalosis
Reduced pH Reduced pH
Respiratory
Metabolic Respiratory Metabolic
Two types
Two types Hypervenytilation Vomiting
Compensated Uncompensated Elevated Anion Gap Normal Anion Gap*

Chronic Acute Diabetes Diarrhoea
Fig. 3.9 Alteration in physiological pH and their biochemical relationship. *Anion gap 5 [Na1] − [(Cl−) 1 (HCO3 −)].
Description of alteration in the pH

As discussed above, the pH of blood is maintained constant by the physiological efforts made by kidney and lungs as well as
fine tuning is performed by carbonate, phosphate or protein buffers. Any physiological or metabolic abnormality may lead to
the change in pH of blood beyond the buffering capacity of existing buffers, these conditions are called as acid-base disorders
in clinical terms. Clinically, acid base disorders are categorized into respiratory acid base disorders and metabolic acid base
disorders. We will now discuss each of these disorders in detail. Refer Fig. 3.9 for outline of types of acid-base disorders.
A. Respiratory acid-base disorders

In respiratory acid–base disorders, the primary disturbance is caused by changes in arterial blood pCO2. Respiratory disorders
are related to changes either in the amount of air moving in or moving out of the lungs (ventilation), or in the ability of gases to
diffuse across the alveolar membrane (gas exchange). In both cases pCO2 changes and the carbonic acid concentration rises or
falls. These disorders are of two types, alkalosis and acidosis.
a. Respiratory Acidosis
Respiratory acidosis may be acute or chronic. Acute conditions occur within minutes or hours. They are uncompensated. Renal
compensation has no time to develop as the mechanisms that adjust bicarbonate reabsorption take 48–72 hours to become
fully effective. The primary problem in acute respiratory acidosis is alveolar hypoventilation. If airflow is completely or partially
reduced, the pCO2 in the blood will rise immediately and the [H1] will rise quickly. A resulting low pO2 and high pCO2 causes
coma. If this is not relieved rapidly, death results. Examples of acute (uncompensated, respiratory acidosis) are choking,
bronchopneumonia and acute exacerbation of asthma/COAD.
Chronic respiratory acidosis usually results from chronic obstructive airways disease (COAD) and is usually a longstanding
condition, accompanied by maximal renal compensation. In a chronic respiratory acidosis, the primary problem again is usually
impaired alveolar ventilation, but renal compensation contributes markedly to the acid–base picture. Compensation may be
partial or complete.
The kidney increases Hydrogen ion excretion and ECF bicarbonate levels rise. Blood [H1] tends back towards normal. It takes
some time for the kidneys to respond to a high PCO2 and a high [H1], and therefore compensation will only be maximal some days
after the onset of the clinical problem. In many patients with chronic respiratory conditions, extensive renal compensation will
keep the blood [H1] near normal, despite grossly impaired ventilation. Examples- chronic bronchitis and emphysema.
53
b. Respiratory Alkalosis
Respiratory alkalosis is much less common than acidosis but can occur when respiration is stimulated or is no longer subject to
feedback control. Usually these are acute conditions, and there is no renal compensation. The treatment is to inhibit or remove
the cause of the hyperventilation, and the acid–base balance should return to normal. Examples are: hysterical over breathing,
mechanical over-ventilation in an intensive care patient and raised intracranial pressure, or hypoxia, both of which may stimulate
the respiratory centre.
B. Metabolic acid-base disorders

Metabolic acid–base disorders are caused by an increase in H1 production or a loss of H1 triggering compensatory mechanisms
that result in the loss or gain of HCO3 –, Direct loss or gain of HCO− will also cause metabolic acid base disorders. Primary metabolic
acid– base disorders are recognized by inspecting the bicarbonate concentration. Respiratory compensation takes place quickly
so patients with metabolic acid–base disorders will usually show some change in blood pCO2 because of hyperventilation or
hypoventilation.
b. Metabolic acidosis
In a metabolic acidosis, the primary problem is a reduction in the bicarbonate concentration of the extracellular fluid. Example of
metabolic acidosis are:
•• increased production of hydrogen ions and ingestion of hydrogen ions, or of drugs that are metabolized to acids
•• impaired excretion of hydrogen ions by the kidneys and loss of bicarbonate from the gastrointestinal tract or in the urine
Metabolic acidosis can be of two types one that affects the anion gap and other that does not affect the anion gap [Note: Anion
gap 5 [Na1] − [(Cl−) 1 (HCO3 −)] In a healthy person, the anion gap has a value of between 6 and 18 mmol/L.]
Examples of metabolic acidosis with elevated anion gap

•• Renal disease. Hydrogen ions are retained along with anions such as sulphate and phosphate.
•• Diabetic ketoacidosis. Altered metabolism of fatty acids, as a consequence of the lack of insulin, causes endogenous
production of acetoacetic and b-hydroxybutyric acids.
•• Lactic acidosis. This results from a number of causes, particularly tissue anoxia. In acute hypoxic states such as respiratory
failure or cardiac arrest, lactic acidosis develops within minutes and is life-threatening.
•• Certain cases of over dosage or poisoning. The mechanism common
to all of these is the production of acid metabolites. Examples
include salicylate overdose Clinical Note
It is not uncommon for patients to have
Examples of metabolic acidosis with normal anion gap
more than one acid–base disorder. A patient
(hyperchloraemic acidosis) may have both a metabolic and respiratory
•• Chronic diarrhoea or intestinal fistula. Fluids containing bicarbonate acidosis, such as the chronic bronchitis patient
are lost from the body. who develops renal impairment. In such a
•• Renal tubular acidosis. Renal tubular cells are unable to excrete patient with a raised [H1], the PCO2 will be
hydrogen ions efficiently, and bicarbonate is lost in the urine. increased and the bicarbonate concentration
will be low, both expected findings in primary
respiratory and primary metabolic acidosis.
a. Metabolic alkalosis
Examples of mixed acid-base disorders include
Metabolic alkalosis means a decrease in the H1 ions in the blood and patient with chronic obstructive airways
extracellular fluids due to excessive loss of hydronium ions. The condition disease, hyperventilation salicylate poisoning
that may cause the metabolic alkalosis are: in which respiratory alkalosis occurs due to
stimulation of the respiratory centre, together
•• Loss of hydrogen ion in gastric fluid during vomiting. This is
with metabolic acidosis due to the effects of
especially seen when there is pyloric stenosis preventing parallel the drug on metabolism.
loss of bicarbonate-rich secretions from the duodenum.
54
Chapter 4: Bioenergetics and Energy Coupling
Condition Formula Use in Biochemistry Caution while solving
Gibbs free energy for ion DG 5 DG°1 2.303 RT log Determination of spontenuity of Unit of G in Joules, value of
transport (osmotic1Donnan) [D]/[O]1 nFψ movement of molecules across plasma R 5 8.31, T 5 in Kelvin, n is
ψ is membrane potential membrane movement (osmotic and no. of electrons involved, E in
donnan, i.e. molecules are charged ) or to volts. F 5 96500
n 5 charge on ion that is
calculate concentrations across plasma
transported
membrane
[D] 5 concentration in destination compartment, [O] concentration of ions in origin compartment, R 5 gas constant (8.31), F 5 faraday
constant (96500), T 5 Absolute temperature, K 5 Equilibrium constant
Note that if we use energy terms in calories, gas constant R 5 2 and faraday constant: 23,062 should be used
Also, the change or potential may be represented as V 5 1.602176565 3 10 –19 3 eV / C
4.3 Some examples of Gibbs law in Biological systems

Law of spontaneity is strictly applicable to the biochemical reactions therefore using the classical Gibbs relation one can easily
predict the spontaneity of a reaction. Also, the additional relationships between the equilibrium constant, and standard reduction
potential help biologists to calculate the Gibbs free energy or predicting spontaneity of a reaction. In order to illustrate all possible
type of examples the examples have been categorised into following sub-sections a. bioenergetics of mitochondrial – that
primarily represents redox reactions of ATP generation/hydrolysis. b. bioenergetics of Chloroplast – that primarily deals with
bioenergetics of redox equivalents; and c. bioenergetics of membrane transport – where the osmotic and Donnan equilibrium
based problems have been discussed.
4.3.1. Bioenergetics of Mitochondria

Mitochondria is the powerhouse of cell and responsible for generation of most of the energy equivalents. The primary and most
occurring bioenergetics event that occur in mitochondria is ATP generation, which is later hydrolysed for various cellular functions.
Primarily, the generation of ATP is a process of sequential transfer of electron from a molecule of high reduction potential to low
reduction potential. [It is important to recall that standard reduction potential means the potential of electron donation reaction
with respect to standard hydrogen electrode] If a molecule has high reduction potential it is capable of undergoing reduction
(gain of electron), and if a molecule has high oxidation potential it is capable of undergoing oxidation (loss of electron). Electron
transport chain begins with oxidation of NADH to NAD1 which an oxidation reaction (and for this reason formation of ATP by
this process is called oxidative phosphorylation). Figure below describes how the potential determines the flow of electron from
NADH to terminal acceptor Oxygen by a gradation of oxidation potential (Fig. 4.7).
Energetics of ATP synthesis Energetics of ATP hydrolysis
-4.0
ATP + H2O ADP + Pi NAD+ (-0.32 V)
NADH
Standard free energy change ΔGº = -35 KJ/Mol 2e -
Midpoint redox potential (V)
FAD Succinate
FMN (-0.03 V) FMNH2 2e- (0.03V)
0.0 2e- Ubiquinone (0.06V) FADH2 Fumarate
Fe3+ Cyt b (0.04 V) Fe2+
e-
Fe3+ Cyt c (0.22V) Fe2+

e-
Fe3+ Cyt a (0.22V) Fe2+ Cytochrome
oxidase
Free energy of hydrolysis is variable 0.4 e-
Fe3+ Cyt a3 (0.39V) Fe2+

Ratio of ATP/ADP drives Q
ΔG = ΔGº+ RT ln Q and hence Gibbs free energy 2e-
Hence the ease of hydrolysis of ATP is not similar

at all sub-cellular locations
1O H2O
2 2
0.8 (0.82 V)
Fig. 4.7. Bioenergetics of mitochondria: note that difference in the ATP/ADP ratio regulates the free
energy changes, and redox potential values guide the transport of electron in mitochondrial electron
transport chain.
69
6.3 Detailed description of standard amino acids

Now, we shall discuss the structure and functions of each of the amino acids. During our discussion we will follow the last type of
classification of amino acids which was particularly chosen due to its relevance with structures and their functional relationship.
Following is the description of all the standard amino acids, biological relevance their relation to the structure of their side chains.
1- Glycine: (Gly, G)
•• Simplest amino acid, smallest amino acid, non-chiral so no optical activity.
•• Sweet in taste hence name glycine.
•• In higher eukaryotes, D-Aminolevulinic acid, the key precursor to porphyrins, is biosynthesized
from glycine and succinyl-CoA.
•• Glycine provides the central C2N subunit of all purines, It also acts as inhibitory neurotransmitter that act in spinal cord.
(Refer Chapter 13; introduction to metabolism to find which part of purine is contributed by glycine).
2-Alanine (Ala, A)
•• Performs the function of removal of pyruvate and glutamate from muscles (Glutamate gives NH2
group to pyruvate forming alanine and alpha ketoglutarate, which is transported to liver) thus
reducing the energy load on muscles.
•• Carries ammonia from muscles to the liver (Glucose- Alanine cycle).
3. Valine (Val, V)
•• Isolated from valeria herb hence named, valine.
•• It is one of the branched chain amino acid.
•• Replaces glutamic acid in Hb causing Sickle cell anemia (hydrophobic nature disrupts the structure).
4. Leucine (Leu, L)
•• Major component of the subunits in ferritin, astacin.
•• It is also a branched chain amino acid.
•• Leucine potently activates mTOR i.e. the mammalian target of rapamycin kinase that regulates cell
growth.
•• Important promoter of muscle development and prevents muscle damage.
5. Isoleucine (Ile, I)
•• Another branched chain amino acid.
•• Biotin, sometimes referred to as Vitamin B7 or Vitamin H, is an absolute requirement for the full
catabolism of isoleucine (as well as leucine).
Clinical Note
Maple syrup Urine (MSUD) is a metabolic disorder caused by a deficiency of the
branched-chain alpha-keto acid dehydrogenase complex (BCKDC), leading to a buildup of
the branched-chain amino acids (leucine, isoleucine, and valine) and their toxic by-products
(ketoacidosis) in the blood and urine. Death may occur due to swelling of brain and
seizures. It is also called as branched chain keto acidosis. It follows autosomal recessive
inheritance pattern.
100
Chapter 6: Amino acids: Structure and Properties
6. Methionine: (Meth, M)
•• This amino-acid is coded by the initiation codon AUG which indicates mRNA’s coding region where
translation into protein begins.
•• One of the two sulphur containing amino acids other being Cysteine.
•• Methionine is one of only two amino acids encoded by a single codon (AUG) in the standard genetic
code(tryptophan, encoded by UGG, is the other).
•• Its derivative S-adenosyl methionine (SAM) serves as a methyl donor in metabolic reactions..
•• Improper conversion of methionine can lead to atherosclerosis.
•• Methionine is needed to make creatine.
7. Proline; (Pro, P)
•• Only amino acid that contains secondary amino group/ Imino group.
•• L-Proline is an osmoprotectant and therefore is used in many pharmaceutical, biotechnological applications.
•• In brewing, proteins rich in proline combine with polyphenols to produce haze (turbidity)
•• Proline is the only amino acid that does not form a blue/purple colour with ninhydrin.
•• With ninhydrin, (used in chromatography) Proline, instead, produces an orange/yellow colour; other
amino acids give purple colour.
•• Mostly found in turns of the beta helices, as it has restricted set of torsion angles regions.
8. Tryptophan (Trp, W)
•• Plants and microorganisms commonly synthesize tryptophan from shikimic acid or anthranilate.
•• Serotonin (a neurotransmitter), synthesized via tryptophan hydroxylase. Serotonin, in turn, can
be converted to melatonin (a neurohormone), via N-acetyltransferase and 5-hydroxyindole-O-
methyltransferase activities.
•• Niacin (vitamin) is synthesized from tryptophan via kynurenine and quinolinic acids as key
biosynthetic intermediates.
•• When sieve tube elements in plants undergo apoptosis tryptophan is converted to auxins (a
phytohormone).
9. Tyrosine (Tyr, Y)
•• The word “tyrosine” is from the Greek ‘tyri’, meaning cheese, as it was first discovered in 1846 by
German chemist Justus von Liebig in the protein casein from cheese.
•• It functions as a receiver of phosphate groups that are transferred by way of protein kinases
(so-called receptor tyrosine kinases).
•• A tyrosine residue also plays an important role in photosynthesis. In chloroplasts (photosystem II), it
acts as an electron donor in the reduction of oxidized chlorophyll i.e. light reactions.
10. Phenylalanine (Phe, F)

•• Phenylalanine is a precursor for tyrosine, the monoamine signalling molecules dopamine,
norepinephrine (noradrenaline), and epinephrine (adrenaline), and the skin pigment melanin.
•• Sold as a nutritional supplement for its reputed analgesic and antidepressant effects.
•• Due to its hydrophobicity, phenylalanine is nearly always found buried within a protein. The p
electrons of the phenyl ring can stack with other aromatic systems and often do within folded
proteins, adding to the stability of the structure.
101
7.4.2 Primary Conformation

The primary structure of protein or peptides is essentially made up of covalently linked amino acids which do not have any
other secondary modifications or interactions. We may say that the basic composition of the backbone of protein is called as its
primary structure. Any alteration in the primary structure itself may be severely affecting the higher orders. In a simple example
of sickle cell anemia where valine is replaced by glutamine (V6Q) proves to be deteriorating for hemoglobin’s gross structure and
its function.
Note: Generally proteins are not observed in primary level of structure in native form but remain folded, however addition of mild
amount of detergents or heating; may render the proteins in primary conformations.
7.4.3 Secondary Conformations

Secondary structures are defined as protein conformations that are formed due to folding of peptide backbone; this folding is
caused primarily by carbonyl group and amino group present in each peptide linkage which are capable of interacting by folding
of backbone. When it comes to secondary conformation we often imagine about alpha helices and beta sheets as predominant
forms of secondary structures but as a matter of fact radom coils are one of the most abundant type of secondary structures. Let
us discuss the details of the three types of secondary structures, Helices, Sheets and Random coils. Apart from these three
major types of secondary structures turns and loops are also minor classes of the secondary structures. Some biochemists also
classify the secondary structures as repetitive and non-repetitive. Repetitive include various helical and beta sheet types while
non- repetitive structures are random coils, loops and turns.
Online Support
You tube channel: video 7.3: various secondary conformations
A. HELICES
The simplest structure that a polypeptide can assume with its rigid peptide bonds (and other flexible bonds) is helical structure.
Several terms are used to explain the nature of a helix. The alpha helix was discovered using model building.
•• Pitch (p): Distance of helix along its axis per turn, also called helical rise
•• Turn (t): one complete round of the helix or simple “one helix” is called a turn
•• Number of residues (n) per turn: it is amino acid present in one turn of the helix, values for n are mentioned negative
if helix is left handed
•• Helical rise per turn: p/n
Types of helices
As discussed above, a secondary structure is formed by the interaction of carbonyl group and amino group of the backbone, while
forming a helix, a peptide may have several possibilities for making such interactions. This will be much clear from the Figure 7.8 which
illustrates different possibilities of the hydrogen bond formation within backbone and therefore, different type of helices originate.
The name of different helices are based on the fact that how many residues are present in one complete turn and how many atoms
are present between successive hydrogen bonds (Xm : X is no. of residue, m 5 no of atoms between two hydrogen bonds).
2.2 7 ribbon has strongly forbidden conformation angles, has never been observed in biological systems. As the name suggests
it has only 2.2 residues in one turn and 7 atoms between successive hydrogen bonds. This is much stretched conformation.
310 helix: most often occurs as a single-turn transition between one end of an alpha helix and the adjoining portion of a
polypeptide chain. The longest helix is known to be 15 residues long.
Alpha helix: For a polypeptide made from L-a amino acid residues, the a helix is right handed with torsion angles –57°(phi)
and –47° (psi) n 5 3.6 residues per turn, and a pitch of 5.4 Å. (An a helix of D-amino acid residues is the mirror image of
120
Chapter 7: Proteins and their Conformations
that made from L-amino acid residues: It is left handed with conformation angles 157° 147°, and n 5 –3.6 but with
the same value of p.) alpha helix may be small upto 12 residues or as large as 140 residues. This helix is also called 3.613 helix
P(pi) helix (4.416 helix): This is most compressed conformation of helix, which has highest number of residues per turn. There
are 4.4 residues per turn and 16 atoms between successive hydrogen bonds. This secondary structure has a mildly forbidden
conformation and it has been rarely observed.
O 1
2 16
13
C N 5
7
8 9
10
11 12 14 15 17 18
Cα 3 4 17
H 7 10 13 16
2.27 310 5.117

3.613 4.416
helix helix a helix γ helix
p helix
4.8 A
4.8 A
5.4 A
6A
5.6 A
? 4.6 A 5.6 A
3.8 A 5.6 A
© 2016, Aditya Arya, all rights reserved
Fig. 7.9. Various types of helices. Note the order of the no. of atoms per helix in different variants (2.2 7 → 310 → a → P).
self-test
Q: How do you think polyproline and Polyglcine would fold?
[Hint: polyproline under suitable folding condition forms left handed helix with 3 residues per turn and pitch of 9.4 A
Glycine unexpedtly forms a similar kind of helix just like proline, see high yielding facts for more on polyproline helix]
Conditions that distort alpha helix:

1. Presence of consecutive amino acids that contain bulky side chains (isoleucine, leucine, tryptophan) However, hydrophobic
amino acids at every third position stabilize by hydrophobic interactions.
2. Presence of successive 1ve or –ve amino acids (K, R, H & D, E) causes repulsion and therefore destabilize the helix.
However, if alternative arrangement of 1ve and –ve amino acids stabilize the helix by salt bridges)
3. Presence of Glycine or proline (Glycine causes excessive flexibility and proline causes excessive stiffness) distorts the
regular helical structures.
4. Presence of positive charged amino acids at amino terminal side and -ve amino acids at carboxy terminal side (as chain
behaves as dipole and these disrupt the charge).
Helix capping: The presence of certain residues outside of alpha helices or beta sheets may also be nonrandom. For example,
alpha helices are often flanked by residues such as Asn and Gln, whose side chains can fold back to form hydrogen bonds with
one of the four terminal residues of the helix, a phenomenon termed helix capping.
121
Representation of helices in circular projection

A helical wheel is a type of plot or visual representation used to illustrate the properties of alpha helices in proteins. The sequence
of amino acids that make up a helical region of the protein’s secondary structure are plotted in a rotating manner where the angle
of rotation between consecutive amino acids is 100°, so that the final representation looks down the helical axis. This projection
diagram is often called and “Edmondson wheel” after its inventor. Another similar way of representing a helix is in spiral manner,
Wenxiang diagram (infact, this was the first 2D representation of helix), Yet another format is called as helical net which
is generated by opening the cylindrical surface of each helix along a line parallel to the axis and laying the result out vertically.
How to read a helical wheel

We know that a complete helical turn (which means 360° rotation) is obtained within a span of 3.6 residues (and this is the reason
alpha helix is also called as 3.613 helix). Now imagine looking at alpha helix from top. Any helix from the top would appear like
a wheel. But in order to determine a sequence one must note the angles at which subsequent amino acids will be observed. So
from above information we can say that a new amino acid addition to a peptide in alpha helix will take place at 100o (360o divided
by 3.6). Also one should remember that in case of right handed alpha helix, reading will be done clockwise, while in left handed
alpha helix reading will be done anticlockwise. Colour coding of amino acids based on the nature of side chains may help us to
understand the interaction patterns of helix and the external appearance.
Helical Wheel Wenxiang Wheel Helical Net
M
G A
R N A
K
A A N
I
W G A
8 1
Figure 7.10. Circular representation for alpha helical peptide with arbitrary sequence, A right handed helix is read in a clockwise
fashion, colour coding is often to represent the nature of amino acid side chains. Three different types of representations are depicted
in this figure a. helical wheel, b. Wenxiang wheel and c. helical net.
B. BETA SHEETS
Beta pleated sheet. Pleated sheet’s conformation has repeating phi and psi angles that fall in the allowed region of the
Ramachandran diagram and utilizes the full hydrogen bonding capacity of the polypeptide backbone. In beta pleated sheets,
however, hydrogen bonding occurs between neighboring polypeptide or due to extended folding of same peptide over other as
shown in fig. 7.11. In this conformation, successive side chains of a polypeptide chain extend to opposite sides forming a sheet
like structure. However in this sheet the residues are at some angle (like roof of a house /\/\) such sheet is therefore called as
pleated sheet.
Depending upon the orientation of chains beat sheets are grouped into two types, one called parallel beta sheet, where the
orientation of all atoms in both strands is observed in same direction, while other is called antiparallel beta strand where the
orientation of atoms in both strands is opposite.
Some salient features of beta sheets are given below:
•• Parallel beta sheets are less stable than antiparallel beta sheets, possibly because the hydrogen bonds of parallel sheets
are distorted in comparison to those of the antiparallel sheets
•• Beta sheets may have as small as 2 to as many as 22 strands, most common being 6 strands
•• Jack bean protein concavalin A has 7 stranded beta sheets
122
Chapter 7: Proteins and their Conformations
6.4 Å 6.8 Å
Cα O N H C
Fig. 7.11. Parallel and antiparallel beta sheets, note the position of hydrogen atoms, in parallel sheets the hydrogen bonds are oblique
and therefore weaker than antiparallel, which have stronger hydrogen bonds. Each sheet is pleated and has a pitch of 6.4 Å and 6.8 Å
respectively.
•• Beta sheets are common in globular proteins and are often twisted
•• Smaller amino acids Gly, Ala are common in beta sheets
•• The distance between 1st and 3rd alpha carbon atom is 7 Angstrom.
Table 7.1. Summarizes the physical parameters of common secondary structures.
Parameters of repetitive secondary structures.

Structure f y n p(Å) A H-bond (CO,HN)
2.27 helix –78 159 2.2 5.6 7 i, i12

310 -helix –49 –26 3.0 6.0 10 i, i13
Right-handed alpha helix[3.613 helix] –57 –47 3.6 5.4 13 i, i14
pi-helix (4.416) –57 –70 4.4 4.8 16 i, i15
Gamma Helix (g ) NA NA 5.1 4.8 17 i, i16
Parallel beta strand –119 113 2.0 6.4 NA NA

Antiparallel beta strand –139 135 2.0 6.8 NA NA
n is the number of residues per helical turn, p is the helical pitch, and A is the atoms in H-bonded loop.
C. NON REPETITIVE STRUCTURES

By Definition every secondary structure is having hydrogen bonds within the backbone as a common feature, however all
secondary structures do not contain a regular array or shapes which repeat (such as helix or sheets) but often they form
connections between the repeating secondary structures. There are several names given to non-repeated structures based on
their topology.
Random Coil: Random coils are most abundant secondary structures, unlike the name suggests these structures are non-random
in nature, which means a random coil of a protein will have a fixed shape and does not change until conditions are changed,
but the name was based on the fact that they do not follow a pattern. The formation of random coils is also due to interaction
of carbonyl and amino group hydrogen bond formation, however the number of hydrogen bonds are very few due to which
the structure appear loose and hanging in protein structures. [note: random coil is also used for the intermediate unfolded or
misfolded protein structures in various biochemistry book, but the radom coil present in secondary structure is a well-defined
structure and therefore different from unfolded random coil).
123
C A T H Root
Few Secondary structures Mainly alpha Class

α
Structure based
β α+β α/β
Example:
CATH Superfamily 1.10.490.10
Fold Rossaman fold alpha/beta barrel
C -1 : Mainly Alpha Flavodoxin-like
A -10 : Orthogonal bundle
Class T - 490 : Globin like Rubisco
Superfamily TIM
Architechture H - 10 : Haemoglobin
Topology Glycosyltransferase
Homologous
Supefamily Family β-Galactosidase
α-Amylase β-Amylase
Evolution based
Protein acid α-Amylase cyclodextran
Alpha Beta Mainly Beta
glycosyltransferase
Species Aspergillus niger Bacillus circulans
PDB ID 2aaa 2cdg
Fig. 8.1. Two major classification systems for the proteins, CATH and SCOP both use the structural motifs to classify the proteins.
been classified as either alpha keratins, which occur in mammals, or beta keratins, which occur in birds and reptiles. Humans have
over 50 keratin genes that are expressed in a tissue-specific manner. The alpha keratins are part of a broader family of proteins
called Intermediate Filament (IF) proteins.
Structure of alpha keratin is complex and exist as quaternary coiled coil structures, where each right handed alpha helix
twist over other in left handed manner, both the strands are parallel (which means amino terminal of two proteins are on same
side). Each of the two strands of alpha keratin is rich in the hydrophobic residues Ala, Val, Leu, Ile, Met, and Phe. This allows the
hydrophobic core to be formed stabilizing the structure and therefore results in effective coiled coil formation. The helical sense
is right handed with a pitch of 5.4 Å (a helix), but once the dimerization occurs, the pitch of coiled coil is slightly reduced to 5.1 Å.
8.3.1. Types of keratin

There are more than 50 different types of keratin molecules formed in the higher eukaryotes, mostly they are categorized as basic
or acidic. Table 8.1 enlists some of the major types of keratin molecules and their anatomical locations.
Table 8.1. Types of Keratin and their anatomical locations.

Category A (neutral/basic) Category B (Acidic) Anatomical Location
keratin 1, keratin 2 keratin 9, keratin 10 stratum corneum, keratinocytes
keratin 3 keratin 12 Cornea
keratin 4 keratin 13 stratified epithelium
keratin 5 keratin 14, keratin 15 stratified epithelium
keratin 6 keratin 16, keratin 17 squamous epithelium
keratin 7 keratin 19 ductal epithelia
keratin 8 keratin 18, keratin 20 simple epithelium
8.3.2. Interaction in coiled-coil and higher order structures of keratin

The primary structure of keratin consists of seven-residue repeating units in which the first and fourth residues are predominantly
nonpolar. The hydrophobic groups of each keratin subunit form a nonpolar strip that slowly winds down one side of the helix with
136
Chapter 8: Proteins: Classification & model examples
a slight left-handed “roll.” Thus the primary interactions between two polypeptide chains are hydrophobic. Two residues at the
boundary of hydrophobic core form salt bridge and additionally stabilize the coiled coil. Additionally, alpha Keratin is rich in Cys
residues, which form disulfide bonds that crosslink adjacent polypeptide chains. The a keratins are classified as “hard” or “soft”
according to whether they have a high or low sulphur content. Hard keratins, such as those of hair, horn, and nail, are less pliable
than soft keratins, such as those of skin and callus, because the disulfide bonds resist deformation. The cross link between two
chains in coiled coil are illustrated in Fig. 8.2. At the anatomical level, the coiled coils are further organised into higher structures,
4 pairs of such coiled coils wrap together to form a protofilament, four such protofilaments align parallel to each other to form
a microfibril. Cell of various keratinized exoskeleton structures contain millions of such microfibril. The hierarchy of the keratin
protein is shown in Fig. 8.2.
Organization of Keratin into Interactions between two alpha

higher order structure helices to form coiled coil
20- 30 A0 40- 50 A0
Ionic interactions
Protofilament (a pairs of coiled coils)
Microfibril (four protofilaments)
helix 2
Single right handed alpha helix
helix 1
Coiled Coil of two alpha helices
g e
c b Ala
a Lys
d
450 A0
f helix 1 helix 2 f Glu Arg
a d Asp Glu
b g c
e Lys
Hydrophobic interactions
Fig. 8.2. Detailed structure of keratin a twisted coiled coil forming microfibril which finally organise into higher order structures called
microfibrils. Persistent hydrophobic residues help in keratin twisting. General sequence may be represented by a-b-c-d-e-f where a
and f are essentially non-polar moieties.
self-test
Q: Does keratin always have alpha helix, Yes or No, If No give examples of other forms?
[Hint: Birds feathers contain keratin in beta sheets called beta keratin, what happens to the secondary structure if you
stretch the hair. --- it converts from alpha to beta form]
8.4 Silk fibroin (beta keratin)

The silk fibroins produced by insects (silk month) and spiders are often classified as beta-keratins, though it is unclear whether
they are phylogenetically related to vertebrate keratins. Silk in its raw state consists of two main proteins, sericin and fibroin,
fibroin being the structural center of the silk, and sericin being the sticky material surrounding it. Fibroin polypeptide chains are
predominantly in the beta conformation. Its primary structure mainly consists of the recurrent amino acid sequence (Gly-Ser-
Gly-Ala-Gly-Ala)n. The high glycine (and, to a lesser extent, alanine) content allows for tight packing of the sheets, which
contributes to silk’s rigid structure and tensile strength. A combination of stiffness and toughness makes it a material with
applications in several areas, including biomedicine and textile manufacture. Silk does not stretch, because the beta conformation
is already highly extended.
Fibroin is known to arrange itself in three structures, called silk I, II, and III. Silk I is the natural form of fibroin, as emitted from the
Bombyx mori silk glands. Silk II refers to the arrangement of fibroin molecules in spun silk, which has greater strength and is often
used in various commercial applications. Silk III is a newly discovered structure of fibroin and is formed principally in solutions of
fibroin at an interface (i.e. air-water interface, water-oil interface, etc).
137
Similarly, the silk from the spider web also has predominantly beta structures, which are crystallised into 1–10 nm structures,
which further form micelles of 10–100 nm and at macroscopic level liquid crystals of 100-600 nm are formed that appear on the
surface of silk cocoons or spider webs (Fig. 8.3).
Polypeptide β-sheet structure Micelles Liquid Crystals Nanofibrils Spider Web

0
||
HN CH C Cocoons
R
0.1 to1nm 1-10 nm 10-100nm 100-500nm > 10 mm
500- 1000 nm
Fig. 8.3. Hierarchical organization of silk protein in silk cocoons and spider webs. (credit http://www.cell.com/).
8.5 Collagen
Collagen is also a coiled coil with distinct tertiary and quaternary structures. The quaternary structure of collagen is commonly
known as triple helix or sometimes Madras helix (as it was discovered in Madras University by Prof. Ramachandran).
Anatomically, collagen is an important constituent of connective tissue and it glues together various histological structures. For
this same reason this protein was named collagen as collagen in Greek
means glue producing. Clinical Note
Biochemically, collagen is made up of three separate polypeptides, called What causes bleeding gums in
alpha chains. Each chain contains a 500-1000nm
repeating amino acid sequence scurvy? Prolyl hydroxylase is an apoenzyme
that require ascorbate or vitamin C as a
depicted as Gly–X–Y, where X is often Pro, and Y is often 4-Hyp
cofactor. In absence or deficiency of vitamin
(Hydroxy proline). Hydroxyproline is not integrated at the time of protein
C, newly formed collagen could not undergo
synthesis, but a normal proline residue is hydroxylated by enzyme Prolyl hydroxylation of tropocollagen, this causes
hydroxylase. (This means if we feed a rat with C14 labelled hydroxyproline, loosening of individual helix in collagen fibers,
collagen will not be radiolabelled, and however it will be radiolabelled if we and therefore, soft skin exposed to physical
feed rat with C14 labelled proline). Presence of hydroxyproline in collagen stress such as lips and gums become fragile
helps in stabilizing the individual helix by involving interaction of water and begin to bleed.
molecules with the chain. Mechanism of hydroxylation of proline is shown
in Fig. 8.4.
The Pro and 4-Hyp residues permit the sharp twisting of the collagen helix and due to presence of glycine and proline each of
the helix acquires left handed orientation. These three left handed alpha helices are twisted over each other in right-handed
manner, forming the triple helix structure. Each peptide chain in collagen (also called as tropocollagen) has 3.3 residues per turn
and pitch of 9.57 Å which means 2.9 Å rise per residue (unlike 1.5Å rise per residue in alpha helix). Three such tropocollagen units
are twisted side by side with a 40 nm hole in middle. Average weight of each tropocollagen is 285 kDa and is approximately
300 nm long. Dimensions and structure of each fiber of collagen and also its triple helical structure is shown in Fig. 8.4. Collagen
is synthesized as immature polymer inside the cells, called pro-collagen, which is trimmed at ends to form mature collagen
molecule.
8.5.1. Crosslinking in collagen and higher order structures

The triple helix of collagen is primarily stabilised by inter-chain hydrogen bonding between collagen molecules. At anatomical
level the bundles of triple helices are arranged into higher order structures and interconnected by very strong cross linking which
provide strength to these molecules. Random and less frequent lysine residues in the collagen fibers accomplish this cross linking.
138
Why Amylose fold into helical secondary structure and cellulose fold into linear sheets/fibers?
Amylose, composed exclusively of the relatively bent a (1→4) linkages, prefers to adopt a helical conformation, whereas cellulose,
with b (1→4)-glycosidic linkages, can adopt a fully extended conformation with alternating 180° flips of the glucose units. The
hydrogen bonding inherent in such extended structures is responsible for the great strength of tree trunks and other cellulose-
based materials. Fig. 9.14 depicts the orientation of sugar units acquired during two different linkages. We must remember here
that interaction of iodine with starch occurs only due to the presence of helical confirmation of starch and therefore iodine does
not produce blue colour with any other polymer of glucose, despite of same chemical nature of glucose in all polymeric molecules.
Basic Units Secondary Structure Higher order organization
amourphous lamella non reducing end
6 residues per turn Crystalline

OH Iodine molcule lamella
OH O
Amylose
100 nm
OH O HO
OH 10 nm
HO O Amourphous
O lamella
α-1,4-Linked D-glucose units
Crystalline lamella reducing end
Starch helix
OH OH OH Middle lamella
Cellulose
O HO O Pectin
O O O O
HO O HO Cellulose
OH OH OH
Primary cell wall
β-1,4-Linked D-glucose units
Hemi-cellulose
Plasma Membrane Free Proteins
© 2016, Aditya Arya, all rights reserved Linear fibers
Fig. 9.14. Structural organization of amylose and cellulose. Note the binding site of iodine in helical cavity of amylose (at least 6 turns
of helices are require to develop blue colour), one I2 molecule binds per two turns (per 12 residues). Higher order organization of amylose
shows much compact structure, which is observed inside the starch containing food such as wheat and rice. Cellulose form sheet like
structures, which contain perpendicular fibers interwoven on each other forming complex plant cell wall structures.
Hemicellulose consists of b -1,4 linked glucose residues (like cellulose) that are substituted with other sugars; xyloglucan is the
predominant hemicellulose - others include glucuronoxylan, arabinoxylan, glucomannan, and galactomannan. As cells increase
in volume, H-bonds that link cellulose and hemicellulose loosen, allowing the internal osmotic pressure of the cell to push apart
the cellulose microfibers.
Pectin
Pectins are complex acidic polysaccharides that contain 1,4-linked b -D-galactosyluronic acid residues; they resemble
glycosaminoglycan (GAG) chains. Pectins that have been characterized include homogalacturonan (65% of plant pectins),
substituted galacturonans like apiogalacturonan and xylogalacturonan and rhamnogalacturonan I and II embedded within the
cellulose/hemicellulose network. Pectins provide hydration and additional strength to the primary wall. Pectins can be modified
by methyl esterases; extent of methylation can determine porosity and stiffness of cell wall.
Chitin
Chitin is similar to cellulose, both in its biological function and its primary, secondary, and tertiary structure. Chitin is present in
the cell walls of fungi and is the fundamental material in the exoskeletons of crustaceans, insects, and spiders. The structure
of chitin, an extended ribbon, is identical to that of cellulose, except that the –OH group on each C-2 is replaced by –NHCOCH3,
so the repeating units are N-acetyl-D-glucosamines in b (1→4) linkage. Like cellulose, the chains of chitin form extended
162
chap t er
Lipids: Structure and
Biological Functions
10

•• Understanding the structure of lipids
•• Classification of lipids and their biological roles
•• Some physico-chemical properties of lipids
•• Lipid storage disorders
10.1 Introduction Interesting Fact

Lipids are a heterogeneous group of compounds that share a common
hydrophobic feature therefore remain water insoluble. These are oily or Napalm is a flammable liquid used in warfare.
greasy organic substances, relatively insoluble in water and considerably It is a mixture of a gelling agent, and either
soluble in organic solvents like ether, chloroform and benzene. The term petroleum or a similar fuel. It was initially used
‘lipid’ was first used by the German biochemist Bloor in 1943 for a as an incendiary device against buildings and
major class of tissue components and foodstuffs. Lipids can perform a later primarily as an anti-personnel weapon, as it
variety of functions in living organisms, the most prominent function being sticks to skin and causes severe burns when on
fire. Napalm was developed in 1942 in a secret
energy storage (as reserve) and constituent of plasma membranes. Upon laboratory at Harvard University, by a team led by
oxidation in metabolism, yield large amount of energy. Therefore these are chemist Louis Fieser. “Napalm” is a combination
the molecules of choice for metabolic energy storage. Lipids are known of the names of two of the constituents of
to perform several functions such as storage of energy as a major role, the thickening/gelling agent: co-precipitated
formation of several biological structures such as plasma membranes also, aluminium salts of naphthenic and palmitic acids.
in signalling, and assisting metabolism (enzyme cofactors) in cellular and Its first recorded use was in the European theatre
extracellular components. of war during World War II.
10.2 Fatty acids are building blocks of several lipids

Fatty acids are composed of a long hydrocarbon tail and a terminal carboxyl head. The carboxyl group is usually ionized under
physiological conditions. Fatty acids occur in large amounts in biological systems but only rarely in the free, uncomplexed state.
They are typically esterified to glycerol or other backbone structures like sphingosine (an amino alcohol, described later in
this chapter). Most of the fatty acids found in nature have an even number of carbon atoms (usually 14 to 24). Certain marine
organisms, and some plant based lipids, contain substantial amounts of fatty acids with odd numbers of carbon atoms.
self-test
Q: Why did nature chose even-numbered fatty acids and not odd numbered fatty acids for energy storage?
[Hint: See Chapter 14, metabolism of lipids, to find the answer].
173
Section
3
Chapter 13: Global View of Metabolism 239–253
Chapter 14: Metabolism of Biomolecules 254–295
Chapter 15: Enzymes I: Principles of catalysis296–316
Chapter 16: Enzymes II: Kinetics317–333
Chapter 17: Enzymes III: Regulation 334–352
237
238
chap t er
Global View of Metabolism

13

•• Understanding the basics of metabolism
•• Metabolic pathways are compartmentalised
•• Organ specific metabolism
•• How to study metabolism: Tracing pathways
13.1 Introduction
Interesting Fact
The term metabolism is derived from the Greek word “Metabolismos” for
“change”, or “overthrow”. The history of the scientific study of metabolism
The first controlled experiments in human
spans several centuries and has moved from examining whole animals in early metabolism were published by Santorio
studies, to examining individual metabolic reactions in modern biochemistry. Santorio in 1614 in his book Ars de statica
Although the metabolism of living organism is presented by some of the medicina. He described how he weighed
himself before and after eating, sleep, working,
most complex and intermingled networks but for the ease of understanding
sex, fasting, drinking, and excreting. He found that
metabolic pathway may be grouped into three main categories: most of the food he took in was lost through what
he called “insensible perspiration” In the 19th
a. Anabolic pathways are involved in the synthesis of larger and century when studying the fermentation of sugar
more complex compounds from smaller precursors—e.g. the to alcohol by yeast, Louis Pasteur concluded that
synthesis of protein from amino acids and the synthesis of reserves fermentation was catalyzed by substances within
of triacylglycerol and glycogen. Anabolic pathways are endothermic. the yeast cells he called “ferments”. He wrote that
“alcoholic fermentation is an act correlated with
b. Catabolic pathways are involved in the breakdown of larger the life and organization of the yeast cells, not
molecules, commonly involving oxidative reactions; they are usually with the death or putrefaction of the cells.”
exothermic and produce reducing equivalents, which are processed
mainly via the respiratory chain resulting in the production of ATP.
c. Amphibolic /Anapleurotic pathways, which occur at the “crossroads” of metabolism, acting as links between the
anabolic and catabolic pathways, e.g. the citric acid cycle.
Knowledge of normal metabolism is essential for an understanding of abnormalities underlying disease.
Metabolism operating in normal conditions fluctuates during adaptation to periods of starvation, exercise, pregnancy, and
lactation. Whereas, severe deviation in the metabolism resulting from nutritional deficiency, enzyme deficiency, abnormal
secretion of hormones, or the actions of drugs and toxins is a metabolic disorderedness and may also be lethal. On an average
an adult human being requires about 8 3 106 – 107 Joules of energy every day. The energy demands may fluctuate depending on
the activity level of an individual. It has been observed that larger animals require less energy per kg body weight compared to
smaller animals. For human beings this requirement is met from carbohydrates (40–60%), lipids (mainly triacylglycerol, 30–40%),
and protein (10–15%).
239
Precursor Molecules Cell Macromolecules

Amino acids Chemical Energy Used Proteins
Sugars
Fatty acids
Anabolism Polysaccharides
Lipids
Nitrogenous
Metabolism
ATP NAD
NADH NADP
NADPH FAD
FADH2 ADP
End products Macromolecules

Amino acids
Proteins
Sugars
Fatty acids
Nitrogenous Chemical Energy Released
Catabolism Polysaccharides
Lipids
Fig. 13.1. General depiction of type of reactions occurring during metabolism.
The very purpose of metabolism is to provide energy to organisms in order to keep them alive. In strict thermodynamic sense,
we (and infact all living organisms) are highly ordered entities, which are constantly being pulled away towards randomness
(as per the second law of thermodynamics). Hence, it is essential to constantly supply the system with some energy such
that, the stability (ordered state) of the system may be maintained. So, whatever food we take, the fate and outcome of
the net metabolism is to generate free energy to keep organisms in homeostasis and ordered. Else, one would start losing
orderedness and complete loss of orderedness will cause dispersal of atoms apart into the universe which in general terms
called as death.
13.1.1. Basal metabolic rate

Basal Metabolic Rate (BMR) is defined as the amount of energy (in
calories) that a person needs to keep the body functioning at rest. As
we know that during rest several processes such as breathing, blood Trick to Remember
circulation, controlling body temperature, cell growth, brain and nerve
Caloric Balance Equation is the
function, and contraction of muscles are still operational and therefore
mathematical summation of the caloric
BMR is an indicative of energy expenditure trend of an individual. Some intake (1) and energy expenditure (-) from
people quickly gain weight by eating less while many do not gain weight all sources. It is represented as:
even by eating more, this is also directly associated with their Basal
Caloric Balance 5 1 food ingested (kcal)
metabolic rate (BMR). The basal metabolic rate accounts for about 60 - basal or resting metabolic rate (kcal) -
to 75% of the daily calorie expenditure by individuals. BMR can be thermogenesis (kcal) - work or exercise
affected by various factors such as level of exercise, muscle mass, stress metabolism (kcal) - energy excreted in
and age. Measurement of the BMR can be performed experimentally waste products (kcal).
by direct measurement of exhaled gases using indirect calorimetry or
through some of the classically developed mathematical formula which use age, sex, height and weight of an individual.
The liver is the largest consumer of energy at rest (29–32%), followed by the brain (19–21%), muscles (18%), heart (10%), lungs
(9%) and kidneys (7%). Fig. 13.2 depicts the energy expenditure of normal human body.
240
13.4 Metabolic pathways are anatomically heterogeneous

In prokaryotic or unicellular organism the basic metabolism of the cell remains same as higher eukaryotes or multicellular organism
i.e ATP is the energy currency in almost all the cells and generated by similar exergonic reactions. However, in higher organisms
another important aspect is maintenance of constant metabolic flux at histological level depending on the biological roles of the
tissue and therefore local metabolism at the anatomical level varies in multicellular organisms. Following is the glimpse of major
biochemical conversion predominating in different organs.
13.4.1. Metabolism of brain

Glucose is the primary fuel for the brain. Only under prolonged starvation brain utilizes ketone bodies as metabolic fuel. The
brain has no capacity to store fuels and needs a continuous supply of glucose. The primary requirement of energy to the brain
is for maintaining the electrostatic potentials required for nerve impulse transmission (70% of the energy is used to maintain
the Na1, K1 membrane potentials). The average brain consumes 120 g of glucose a day at the rate of around 5 – 6 g per
hour. The brain also synthesizes neurotransmitters and receptors. Glucose is transported into the brain by the GLUT-3 glucose
transporter. This transporter has a Km value of 1.6 mM for glucose. If the blood glucose concentration remains below km value,
the brain becomes dysfunctional leading to coma and brain death. Fatty acids cannot fuel the brain because they cannot traverse
the brain blood barrier. Under prolonged starvation ketone bodies can replace glucose as a fuel for the brain. Glycolysis and Citric
Acid Cycle are the two predominant biochemical pathways in brain. Additionally amino neurotransmitter (such as Glutamate,
Glycine) biosynthesis also occurs in brain. Lactate synthesised in astrocytes via anaerobic respiration of glucose may also be
used by nerve cells for energy generation.
13.4.2 Metabolism of Muscle

The muscles can use glucose, fatty acids and ketone bodies for fuel. Unlike
the brain the muscles have stores of glycogen. This glycogen is readily
Clinical Note
converted into glucose when needed for bursts of activity. ATP can be Creatinine is a waste product of
generated faster by glycolysis than by oxidative phosphorylation. As a creatinine phosphate breakdown. Creatinine
result, the rate of glycolysis far exceeds the maximum rate of the citric acid phosphate is a high-energy compound found in
cycle. When this happens pyruvate is converted into lactate to regenerate skeletal muscle tissue and is released during
muscle breakdown. There is no biological use
NAD1 needed for glycolysis. The accumulation of lactic acid decreases
for creatinine so the kidneys excrete it all.
the pH and reduces the muscles efficiency. The lactate is released by
Creatinine level is a reflection of glomerular
the muscle tissue into the blood where is absorbed by the liver and by filtration and a very good indicator of renal
gluconeogenesis reconverted back into glucose. This interchange is called function. The levels of creatinine is elevated
the Cori cycle. in renal disease and muscle wasting disorders
Another cycle that transports ammonia to liver is called Pyruvate such as muscular dystrophy and acromegaly,
shock, and rhabdomyolysis. High levels of
-Alanine cycle (Fig. 13.4a). This shifts the metabolic burden for the
ascorbic acid and cephalosporin antibiotics
reduction of lactate from the active muscle tissue to the liver. Most of the
may cause a false positive elevation. Treatment
oxygen in the muscles is used for oxidative phosphorylation which in turn is is to restore kidney function or dialysis.
responsible for restoring ATP and phosphocreatine levels. Skeletal muscle Creatinine Clearance Test: Compares the
contains 10-30 mM phosphocreatine. Phosphocreatine is used to rapidly amount of creatinine in the urine to the amount
regenerate ATP from ADP using phosphocreatine kinase. During vigorous in the blood over the same time period. (Male:
muscular work all the phosphocreatine is converted into creatine. During 95 – 135 ml/min Female: 85 – 125ml/min) is
recovery phase creatine is re-phosphorylated to phosphocreatine. an indicative of glomerular filtration rate.
During rest primary source of energy to the muscles are fatty acids which
include the beta oxidation of fatty acids in mitochondria and generation of
ATP, while during mild aerobic exercise the source of energy is glucose and glycogen, while during a sprint the energy source is
glucose (broken down anaerobically), phosphocreatine and ATP. Birds, capable of flying non-stop several thousands of kilometers
store a large amount of fatty acids in the muscles and use them during flight. Time dependent expenditure of various energy
molecules in the muscle is shown in Fig. 13.4b.
244
Chapter 13: Global View of Metabolism
a. Biochemical interaction between liver and muscle b. Key energy sources in working muscles
MUSCLE
LIVER
100 ATP
Phosphocreatine
Percentage of work energy

Glucose
6-phosohate Glucose Glucose
Glycolysis 75
Glycogen Gluconeogenesis
Pyruvate Pyruvate Anarobic Metabolism
Ammonia
50
Lactate Lactate
Alanine Alanine
25
Aerobic Metabolism
Protein 0
Cori Cycle Glucose-Alanine Cycle degradation
0 30 60 90 20
Duration of work (sec.)
Fig. 13.4. Metabolism in Muscles, a. Cori cycle and Glucose-alanine cycle operating between liver and skeletal muscles. b. temporal
distribution of various energy sources in muscles.
13.4.3. Metabolism of cardiac Muscle

The heart muscle always functions aerobically and has virtually no glycogen Clinical Note
reserves. Cardiac muscle have a heavy workload, about 70% of the energy The cardiac muscle membrane is
is expanded in mechanical contraction of ventricle. Cardiac muscles have permeable to adenosine and the release of
unique aerobic respiration characteristics. During rest, 70% of the energy adenosine during ischemia (sudden decrease
requirement of the muscle is met through fatty acids beta oxidation. The in tissue oxygenation) is followed by a net loss
role of glucose is relatively small as energy source. Lactate is also valuable of ATP, about 50% adenosine is lost in 30 min
energy substrate for the heart. Calcium ions inside the cardiac cells stimulate ischemia, while the restoration takes place at
the oxidative phosphorylation and therefore calcium imbalance is associated very slow pace (about 2% per hour). Severe
with contractile abnormalities in heart. About 95% of the energy sources adenosine and ATP depletion during ischemia
are utilsed in generation of ATP which is then used for contractile activity. is one of the major cause of death in ischemic
conditions.
13.4.4. Metabolism of Adipose Tissue

The function of the adipose tissue is to store triacylglycerols and release fatty acids as needed. The liver is the major site of fatty
acid synthesis. These fatty acids are esterified with glycerol to form triacylglycerols and packaged for transport as very Low Density
Lipoproteins (LDL). The adipose tissue absorbs the free fatty acids carried by the very low density lipoproteins by hydrolyzing the
triacylglycerols with extracellular lipoprotein lipases. The lipases are activated by insulin. After the fatty acids have been transported
into the adipose cell, the triacylglycerols are reassembled and stored. Adipose tissue needs a steady supply of glucose to generate
glycerol 3- phosphate needed for triacylglycerol synthesis. When the blood glucose concentration is low, glucagon activates a lipase
which hydrolyzes the triacylglycerol stores in the adipose tissue. The free fatty acids and glycerol are released into the blood stream.
Serum albumin carries the fatty acids to the tissues and the glycerol is absorbed by the liver and used for gluconeogenesis.
13.4.5. Metabolism of Kidney

The major role of the kidney is to produce urine which is a vehicle for excreting water soluble waste products. During excretion it
is critically important to maintain the salt balance or electrolyte balance, this is regulated by selective reabsorption and secretion
of ions in the kidney (readers are suggested to consult standard physiology text for details). Regulation of electrolyte balance in the
kidneys is further regulated by specialized amino acid metabolism and carbohydrate metabolism in kidney. Most important reaction
in renal metabolism is conversion of Glutamine to Glutamate by enzyme glutaminase, during this reaction ammonia is also generated.
This excretion of ammonium ions is an important mechanism of renal acid-base regulation. During chronic acidosis, glutaminase is
induced in the kidney, which leads to an increase in the amount of ammonium ions excreted. Glutaminase can also be found in the
245
Chapter 14: Metabolism of Biomolecules
CARBOHYDRATE METABOLISM
Starch Cellulose
Maltose
1 Lactose Sucrose
Digestion in mouth (20-40%)
Enzyme : Salivary amylase
Cellulase (NOT in Humans)

Amylase
Maltase
Lactase
Sucrase
2
Digestion in stomach
Enzyme : none
Acid hydrolysis by HCl
Glucose
3
Digestion in duodenums (50-80%) Glucose
Enzyme: amylase
Glucose
Galactose Fructose
Glucose
Glucose
Digestion in illeum (~100%)
4 5
Enzyme maltase, lactase,
sucrase, dextrinase
Absorption in illeum
Villi through villi, via specific tranporters
into the adjoining capillaries
serosa Jejunal mucosal cell

Pentoses Pentoses
Capillary in villi
Lumen Fructose
GLUT5
villi- capillaries GLUT2
Glucose/Galactose SGLT1
K
+
Na +
Na +
All monosaccharides are Na+ Na+/K+ Pump
6 transported to liver via hepatic portal

system followed by metabolic conversion
or transport to various organs
ATP
Glycogenolysis
Glucose
Glycogen
7
Various metabolic pathways
Gluconeogenesis
Glycogenesis at sub-cellular level

and generation of energy (ATP)
Glycolysis
and other useful metabolites

Ribose
Pentose Phpsphate
Pathway Citric Acid Cycle
H2O Electron Transport Chain

Pyruvate Acetyl coA
CO2 ATPs
Fermentation NADH
Fatty acid biosynthesis
Lactate Fatty Acids
© 2016. Aditya Arya, all rights reserved
Fig. 14.2. Overview of carbohydrate metabolism in human.
257
Chapter 16: Enzymes II: Kinetics
D. CPS 1 and CSP 2

Carbamoyl phosphate synthetase catalyzes the ATP-dependent synthesis of carbamoyl phosphate from glutamine/ammonia
and bicarbonate. This enzyme exist in two different structural variants/ isoforms called carbamoyl phosphate synthase
1 (CPS1) and CPS2. Carbamoyl phosphate synthetase I (CPS1 or CPSI) transfers an ammonia molecule from glutamine or
glutamate to a molecule of bicarbonate that has been phosphorylated by a molecule of ATP. This is involved in the urea cycle
and localised in mitochondria. While CPS2 is an enzyme that catalyzes the reactions that produce carbamoyl phosphate in the
cytosol for the pyrimidine biosynthesis. Neither CPSI nor CPSII require biotin as a coenzyme, as seen with most carboxylation
reactions.
16.4. Measures of enzyme efficiency

Enzyme efficiency can be measured in various ways but the usual parameter to observe the efficiency is the factor by which
enzyme increase the speed of reaction of turns over a reactant into product. However, there are other parameter called as
specificity constant. In the following section we will understand the difference between these two parameters.
16.4.1. Turnover number (Kcat)

Turnover number is equivalent to the number of substrate molecules converted to product in a given unit of time on a single
enzyme molecule when the enzyme is saturated with substrate.
An alternative representation of turnover number is Kcat. It is useful to define a more general rate constant, kcat, to describe
the limiting rate of any enzyme-catalyzed reaction at saturation. If the reaction has several steps and one is clearly rate limiting,
kcat is equivalent to the rate constant for that limiting step. For most of the enzymatic reaction Kcat may be same as K 2 (rate
constant for the formation of product).
k
1 k2 ka

E + s  
ES  
EP E + p
k -1 k -2
For this reaction K3 will be the Kcat as breakdown of EP into E and P is the rate limiting step.
If we observe the Table 16.1, we will find that catalase is one the fastest acting enzyme or it is most efficient enzyme. But this
may not be technically correct, as we may not comment on the efficiency of enzyme until we compare the Km (Recall that Km is
inversely proportional to specificity, and higher its more will the reversal of ES complex to E1S). Therefore a better parameter
called specificity constant is used to define efficiency.
Online Support
You tube channel: video 16.3: Why Kcat is not good measure for efficiency.
Table 16.1. Substrates and Kcat values of some common enzymes.

Enzyme Substrate kCAT (S –1)
Catalase H2O2 40,000,000
Carbonic anhydrase HCO3 400,000
Acetylcholinesterase Acetylcholine 14,000
B-Lactamase Benzlpenicillin 2,000
Fumarase Fumarate 800
RecA protein (an ATPase) ATP 0.4
325
16.4.2 Specificity constant (Kcat /Km)

The parameters kcat and Km also allow us to evaluate the kinetic efficiency of enzymes, but either parameter alone is insufficient
for this task. Two enzymes catalyzing different reactions may have the same kcat, yet the rates of the uncatalyzed reactions may
be different and thus the rate enhancements brought about by the enzymes may differ greatly. Experimentally, the Km for an
enzyme tends to be similar to the cellular concentration of its substrate. An enzyme that acts on a substrate present at a very
low concentration in the cell usually has a lower Km than an enzyme that acts on a substrate that is more abundant. The best
way to compare the catalytic efficiencies of different enzymes or the turnover of different substrates by the same enzyme is to
compare the ratio kcat /Km for the two reactions which is called as specificity constant.
Specificity constant 5 kcat /km
Specificity constant as this is obtained by equating in MM equation as below:
k cat
V0 = [E t ][S]
Km
Its units are M –1S –1 (second order rate constant). There is an upper limit to kcat /Km, imposed by the rate at which E and S can
diffuse together in an aqueous solution. This diffusion controlled limit is 108 to 109 M –1s –1, and many enzymes have a kcat /Km near
this range. Such enzymes are said to have achieved catalytic perfection. Note that different values of kcat and Km can produce
the maximum ratio.
Now if we try arranging the top 3 enzymes, first on the basis of turnover number (Kcat) and then on the basis of specificity
constant, we will find a completely reverse order in two different conditions.
Table 16.2. Km, Kcat and Specificity constants of some enzymes.
Enzyme Substrate Kcat (s –1) K m (M) Kcat /K m (M –1S –1)
Acetyl cholinesterase Acetylcholine 1.4 3 10 4 9 3 10 -6 1.6 3 10 8
Carbonic anhydrase C02 1 3 10 6 1.2 3 10 –2 8.3 3 10 7
HCO3 4 3 10 5 2.6 3 10 –2 1.5 3 10 7
Catalase H2O2 4 3 10 7 1.1 3 10 0 4 3 10 7
Crotonase Crotonyl-CpoA 5.7 3 10 3 2 3 10 -5 2.8 3 10 8
Fumarase Fumarate 8 3 10 2 5 3 10 -6 1.6 3 10 8
Malate 9 3 10 2 2.5 3 10 -5 3.6 3 10 7
B-Lactamase Benzylpenicillin 2.0 3 10 3 2 3 10 -6 1 3 10 8
16.4.3. Order of enzyme kinetics

What is the order of enzymatic reactions? There may be many answers to this question depending on the context and time point
at which the enzymatic reaction is being evaluated.
Zero order reaction: Usually when we are observing the kinetics of enzymes in the post stabilization state when the ES
complex has been stabilised, enzyme will follow zero order kinetics. In such condition [S]>>Km, so v5Vmax. This means that the
rate is equal to the maximum velocity and is independent of the substrate concentration. The reaction is zero-order kinetics.
First order reaction: The second condition is When [S]<<Km, then V 5 Vmax [S]/Km. This means that the rate and the substrate
concentration are directly proportional to each other. The reaction is first-order kinetics. Usually the linear part of the MM curve
will depict the first order reaction.
Second order reaction: In some cases when [S]<< Km and very little ES will be formed and therefore [E] 5 [Et] so placing these
values in MM equation we get V0 5 K 2/Km [E][S] and here the Kcat /km is apparent second order rate constant so reaction will
follow a second order (Fig. 16.7).
326
High Yielding Facts

High Yielding Facts
The mechanism of partially competitive inhibition is similar to that of non-competitive, except that the EIS
complex has catalytic activity, which may be lower or even higher (partially competitive activation) than that of
the enzyme–substrate (ES) complex. This inhibition typically displays a lower Vmax, but an unaffected Km value.
Uncompetitive inhibition occurs when the inhibitor binds only to the enzyme–substrate complex, not to the free
enzyme; the EIS complex is catalytically inactive. This mode of inhibition is rare and causes a decrease in both
Vmax and the Km value.
Product inhibition is often a regulatory feature in metabolism and can be a form of negative feedback.
Slow-tight inhibition occurs when the initial enzyme–inhibitor complex EI undergoes isomerisation to a second
more tightly held complex, EI*, but the overall inhibition process is reversible. This manifests itself as slowly
increasing enzyme inhibition. Under these conditions, traditional Michaelis–Menten kinetics give a false value
for Ki, which is time–dependent. The true value of Ki can be obtained through more complex analysis of the on
(kon) and off (koff) rate constants for inhibitor association.
Paclitaxel (taxol), an organic molecule found in the Pacific yew tree, binds tightly to tubulin dimers and inhibits
their assembly into microtubules in the cytoskeleton.
An example of a neurotoxin are the Glycoalkaloids, from the plant species in the Solanaceae family (includes
potato, tomato and eggplant), that are Acetylcholinesterase inhibitors.
Neurotoxicity can also result from the inhibition of receptors; for example, atropine from deadly nightshade
(Atropa belladonna) that functions as a competitive antagonist of the muscarinic acetylcholine receptors.
Reversible competitive inhibitors, such as edrophonium, physostigmine, and neostigmine, are used in the
treatment of myasthenia gravis and in anaesthesia.
The organophosphate pesticides such as Malathion, parathion, and chlorpyrifos irreversibly inhibit
acetylcholinesterase.
The herbicide glyphosate is an inhibitor of 3-phosphoshikimate 1-carboxyvinyltransferase, other herbicides,
such as the sulfonylureas inhibit the enzyme acetolactate synthase. Both these enzymes are needed for plants
to make branched-chain amino acids.
352
ap p e nd ix
List of Common
Biochemical Tests
1
Test Detection Reagent Test results Comments
Tests for Amino Acids
Purple color Proline gives yellow
Ninhydrin test All alpha amino acids Ninhydrin
(at pH 4-8) colour
Mechanism: (triketohydrindene hydrate), terminal amines of lysine residues react with ninhydrin
Only Aromatic amino Conc. Nitric Acid Yellow colour
Xanthoproteic test
acids
Mechanism: Yellow colour is due to xanthoproteic acid which is formed due to nitration of amino group.
Pauly’s diazo Test Tryptophan or Histidine sulphanilic acid Red Colour Azo salt formation
Mechanism: Sulphanilic acid upon diazotization in the presence of sodium nitrite and hydrochloric acid results in the formation a diazonium salt
which couples with either tyrosine or histidine in alkaline medium to give a red coloured chromogen (azo dye).
Phenolic amino acids Mercuric sulphate in Red Colour
Millon’s test
(Tyrosine) sulphuric acid.
Mechanism: he red colour is probably due to a mercury salt of nitrated tyrosine.
Bromination of histidine in Bromine water Blue/Violet
Histidine test
acid solution
Mechanism: This reaction involves bromination of histidine in acid solution, followed by neutralization of the acid with excess of ammonia.
Heating of alkaline solution develops a blue or violet coloration.
Only for tryptophan glyoxilic acid 1 sulphuric Purple
Hopkins cole test
acid
Mechanism: The indole moiety of tryptophan reacts with glyoxilic acid in the presence of concentrated sulphuric acid to give a purple colored product.
Sakaguchi test
Arginine or (Guanidine a - naphthol Red Colour Under alkaline condition
cmpond)
Mechanism: Under alkaline condition, a - naphthol (1-hydroxy naphthalene) reacts with a mono-substituted guanidine compound like arginine,
which upon treatment with hypobromite or hypochlorite, produces a characteristic red color.
boiling with sodium lead acetate
Lead sulphide test
hydroxide (hot alkali)
Mechanism: reaction is due to partial conversion of the organic sulphur to inorganic sulphide, which can detected by precipitating it to lead
sulphide, using lead acetate solution.
Imino acids such as isatin reagent blue colour
Folin’s McCarthy Test
Proline and HyPro
Mechanism: Proline and hydroxyproline condense with isatin reagent under alkaline condition to yield blue colored adduct.
Sullivan test Cysteine and Cystine Sullivan reagent red colour
Mechanism: Sullivan reagent (1,2, naphthoquinone-4-sulphonate) reacts with thiol group in presence of Na2S2O4, gives red coloured complex.
isatin reagent under
Isatin test Proline and hydroxyproline Blue Colour Also detects methionine.
alkaline condition
Mechanism: Imino acids such as Proline and hydroxyproline condense with isatin reagent under alkaline condition to yield blue colored adduct.
353
ap p e nd ix
List of Common Inhibitors

2
CYTOSKELETAL & CELL DIVISION INHIBITORS
Sr. No. Inhibitor Mode of Action Additional comments
Binds plus end of Actin filament and Prevent

1. CYTOCHALASIN- D
Elongation
Binds G Actin monomers and prevent them from

2. LATRUNCULIN All of these inhibitors can prevent contractile
polymerizing into filament.
ring formation at the time of cytokinesis as
Binds tightly all along the sides of actin filament and Actin is a major component of contractile ring
3. PHALLOIDIN
stabilizes them against depolymerization. in animal cells
4. JASPLKINOLIDE Induces Actin polymerization and stabilizes F actin.
5. SWINHOLIDE Severs filament
Naturally occurs in animal cells to regulate

6. THYMOSIN Actin monomer binding protein in mammalian cells.
cytoskeketan
Binds to Tubulin subunit and prevent their Obtained from the bark of Chinchona, inhibit
7. COLCHICINE
polymerization spindle formation
8. NOCODAZOLE Causes microtubule to depolarize to Tubulin subunit
Binds to Tubulin subunit and prevent their

9. COLCEMID
polymerization
Binds and stabilize microtubules, preventing their Obtained from Yew tree, inhibit retraction of
10. TAXOL
depolymerization spindles
11. VINBLASTINE Binds to beta Tubulin to prevent polymerization
12. VINCRISTINE Binds free Tubulin to prevent Polymerization
MEMBRANE TRANSPORT INHIBITORS

Sr. No. Inhibitor Source Mode of Action Additional comments
1. OUABIN Digitalis purpura Inhibit Na-K pump by binding on K binding site Used as cardiotonic steroid as
they also inhibit Na-K pump to
2. DIGITOXIGENIN Digitalis purpura Inhibit Na-K pump by binding on K binding site increase which in turn block
Na-Ca antiport and elevate Ca in
3. PALYTOXIN Coral Inhibit Na-K pump heart and improve efficiency.
Puffer fish is also called as swell

4. TETRODOTOXIN Puffer fish Block Na channel
fish or blow fish
Inhibit voltage gated Na

5. SAXITOXIN Gonyaulax
Channel
6. VERATRIDINE Sabadilla Bind to Na channel and leave it permanently open
357
Sr. No. Inhibitor Source Mode of Action Additional comments
7. BATRACHOTOXIN Phyllobates auro. Bind to Na channel and leave it permanently open A kind of Colombian frog.
8. BUNGAROTOXIN Krait Inhibits Acetylcholine esterase enzyme
Contains phospholipase A2
9. COBRA TOXIN Cobra
And therefore cause plasma membrane rupture
10. SARIN Chemical synthesis Inhibits Acetylcholine esterase enzyme
Used for the treatment of

Bind to muscarintic type of Ach receptor (inhibitory
11. ATROPINE Atropa belladonna toxicity
Ach receptor)
Used for dialation of Pupil
ELECTRON TRANSPORT CHAIN INHIBITORS

Site of action Compounds Effect on oxidative phosphorylation Use
Rotenone Prevents the transfer of electrons from complex I to ubiquinone by
Pesticide
blocking the ubiquinone-binding site
Amytal a barbiturate that inhibits the electron transport of complex I. Sedative drug
Complex I
Demerol Painkiller that also inhibits complex I.
Piericidin A Inhibits electron transfer; it competes with QB for binding Antibiotic (from
Piericidin A
site in Photosystem II. Streptomyces sp.)
2- Thenoyltrifluoroacetone TTFA binds at the quinone reduction site in Complex II, preventing
(TFFA) ubiquinone from binding.
Complex II Carboxin inhibits oxidation of succinate Fungicide
Malonate and Competitive inhibitors of succinate dehydrogenase (complex II)
Poisons
oxaloacetate
Binds to the Qi site of cytochrome c reductase, thereby inhibiting the
Antimycin A1 Pesticide
oxidation of ubiquinol.
Inhibitor of the quinol oxidation (Qo) site of the cytochrome bc1
Stigmatellin complex in mitochondria and the cytochrome b6f complex of thylakoid Poison
Complex III
membranes.
Myxothiazol Competitive inhibitor of ubiquinol, and binds at the quinol oxidation (Qo)
site of the bc1 complex, blocking electron transfer to the Rieske iron- Poison
sulfur protein.
Cyanide Inhibit the electron transport chain by binding more strongly than oxygen
Carbon monoxide to the Fe–Cu center in cytochrome c oxidase, preventing the reduction
Complex IV Azide of oxygen. [Axide and cyanide bind to the iron when the iron is in the Poisons
Hydrogen sulfide ferric state. Carbon Monoxide binds to the iron when it is in the ferrous
state.]
Oligomycin Inhibits ATP synthase by blocking the flow of protons through the Fo
Antibiotic
subunit.[86]
ATP Synthase
Diclyclohexylcarbodiimide
(DCCD)
2,4-Dinitrophenol, dicumarol All have hydrophobic character making them soluble in the bilipid
carbonyl cyanidep- membrane. All of these decouplers also have dissociable protons
Poisons, weight-
trifluorocarbonyl-cyanide allowing them to carry protons from the intermembrane space to the
loss
methoxyphenyl hydrazone matrix which collapses the pH gradient. The potential energy of the
UNCOPLERS (FCCP) proton gradient is lost as heat.
UCP 1 known as thermogenin (this is one of the 5 types of naturally occurring
uncouplers in mammalian body (present within adipocytes, giving them
brown color, hence the fat containing UCP 1 is also called as Brown fat)
358
Appendix 2: List of Common Inhibitors 359
DNA REPLICATION INHIBITORS

1. MITOMYCINS causes cross-linking between complementary antitumor and antibacterial agents

strands of DNA
2. ACTINOMYCIN D Binds to DNA by intercalating between base

pairs
3. QUINOLONES AND Block DNA gyrase Act against both gram positive and gram
FLUOROQUINOLONES negative
(e.g. CIPROFLOXACIN)
4. COUMERMYCIN,NALIDIXIC Interferes with breakage and rejoining of The gyrA or “swivelase” component of DNA
ACID, OXOLINIC ACID DNA chain hence block DNA gyrase gyrase is sensitive to the action of nalidixic
and oxolinic acids
5. AMPHIDICOLIN Inhibitor of DNA polymerase Inhibit only mammalian nuclear polymerase.
6. NOVOBIOSIN Block binding of ATP to DNA gyrase
TRANSCRIPTION INHIBITORS
1. STREPTOLYDIGIN inhibits transcription Inhibit chain elongation as well as
the initiation process in vitro. In vivo,
by binding to the b subunit
streptolydigin appears to accelerate the
termination of RNA chains
2. RIFAMPICIN (RIFAMYCIN) Binds to the b subunit of bacterial RNA used to treat tuberculosis which is resistant to
polymerase. also inhibits mitochondrial RNA most other antibiotics
polymerase
3. ALPHA AMINITIN Inhibit RNA polymerase II at low concentration Eukaryotic inhibitor, isolated from Amita
(RNA I is completely resistant) phalloides
4. CORDYCEPIN 3’deoxy adenosine inhibit transcription
elongation
5. ACTINOMYCIN D Inhibit the transcription of r-RNA selectively Phenoxazane ring intercalate between
as r-RNA is GC rich. adjacent G:C
6. POLYAMINES(SPERMIDINE, Spicing inhibitors
SPERMIE,PUTRISINE)
PROTEIN SYNTHESIS INHIBITORS

1. STREPTOMYCIN Binds to 30S subunit and cause misreading In prokaryotes
of m-RNA and affects initiation Broad (gram 1, –; mycobacteria)
2. KANAMYCIN, NEOMYCIN, Bind to 30S subunit of ribosome and block In prokaryotes
GENTAMYCIN initiation
3. TETRACYCLINE Bind to 30S subunit and interfere with Broad (gram 1, –; rickettsia and chalymidia
aminoacyl –t- RNA binding.
4. ERYTHROMYCIN Binds to 50S subunit and inhibits In prokaryotes
AZITHROMYCINS translocase. (This is true for mitochondrial ribosomes too)
CLARITHOMYCINS Narrow (gram 1, mycoplasma
DIRITHOMYCIN
359
ap p e nd ix
Reference values in
Blood Tests
3
CBC COUNT CD4 550 – 1600
RBC 4.6-6.0 M/UL Male CD8 200 – 700
4.0-5.4 M/UL Female Wright Stains No Neutrophils
WBC 4.5-11.5 K/uL No Eosinophils
HGB 14.0-18.0 g/dL Male ANC 2.3-8.6 K/uL
12.0-15.0 g/dL Female
HCT 40-54% Male Body Fluids:
35-49% Female CSF
MCV 80-94 fL Male Colorless
80-96 fL Female 0-5/uL WBC
MCH 26-32 pg 63-77% Lymphocytes
MCHC 32-36 g/dL 3-37% Monocytes
RDW 12.5 – 14.5% 0-2% Neutrophils
PLT 150 - 450 K/uL SYNOVIAL
MPV 7.4 – 10.4 fL Colorless/Pale Yellow
<200/uL WBC
DIFFERENTIAL 0-25 %Neutrophils
Lymphocytes 18 – 42% 0-78 % Lymphocytes
Monocytes 2 – 11% 0-71% Monocytes
Granulocytes 50 – 70% 0-2000/uL RBC
Eosinophils 1 – 3% No Crystals Present
Basophils 0 – 2% PERITONEAL (ASCITES)
Bands 0 – 9% No known normal reference ranges
Blasts 0% PLEURAL/ PERICARDIAL
No known normal reference ranges
MISCELLANEOUS For detailed description of reference values visit:
Absolute Eos 0 – .450 K/uL https://en.wikipedia.org/wiki/Reference_ranges_for_blood_
Reticulocytes 0.2 – 2.2% tests
Sickle Cell NEG https://labtestsonline.org/understanding/features/ref-ranges/
start/6
G-6-PD 10 - 60 min
Note: these values are from various available literature and
Westergren ESR 0 – 30 mm/hr Male
may not be associated with exact clinical conditions, therefore
0 - 20 mm/hr Female clinical corelation is not recommended.
Viscosity 1.4 – 1.8
Ham’s Test No Lysis
361
Chapter 14
1. https://accesspharmacy.mhmedical.com/Content.aspx?bookid=1366&sectionid=73243421: Text on introduction of metabolism.
2. http://osp.mans.edu.eg/medbiochem_mi/Cources/Biochemistry/2nd_year_medicine/Protein_metabolism/files/
Lecture_01.htm
3. http://intranet.tdmu.edu.ua/data/kafedra/internal/chemistry/classes_stud.../en/med/lik/ptn/2/02.%20Basic%20
principles%20of%20metabolism%20catabolism,%20anabolism..htm
Good link for carbohydrate metabolism and insulin http://users.atw.hu/blp6/BLP6/HTML/C0389780323045827.htm.
5. https://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/glycogen.htm
6. Figure 14.5 the electron transport chain figure was originally created by KEGG developers and they own the copyright,
www.genome.jp (permission to use pending).
7. In Fig. 14.2, 14.13, 14.20 and 14.27 the digestive system of human being was used from public domain but originally
created by Mariana Ruiz Villarreal. The original image is available at https://commons.wikimedia.org/wiki/File:Digestive_
system_without_labels.svg
Chapter 15- 17
1. Nicholas Price and Lewis StevensBugg. Fundamentals of Enzymology, Cell and Molecular Biology of Catalytic Proteins.
Oxford university press: Excellent text on enzymology and its kinetics
2. T. Introduction to Enzyme and Coenzyme Chemistry. Blackwell Science, 2ndedn, 2004
3. Palanivelu, P. Enzymes, Ribozymes and DNAzymes, Twentyfirst Century Publications, Palkalai Nagar, Madurai - 625 021,
2007. Some of the content on history of enzymology was based on his book (In fact I am indebted to him as he was my
Teacher during Masters in Biochemistry)
4. Palmer & Bonner Enzymes, 2nd Edition Biochemistry, Biotechnology, Clinical Chemistry, Elsevier.
5. A deeper insight into the morpheein model of allosteric regulation may found at https://www.ncbi.nlm.nih.gov/
pubmed/16023348
As this is first edition of the book, if some of the text or images are noticed by original contributors which were not
credited authors may be intimated.
366
About The Author
Dr. Aditya Arya
Dr. Arya is a Ph.D. in the area of Nanomedicine from Defence Research and Development
Organization, Delhi. He has published 20+ research papers in peer-reviewed international
journals, four chapters in international books and authored five books. He holds
membership of several international societies in Nanomedicine, Redox Biology and has
received several awards in the domain of life science research. He has been trained at some
of the prestigious institutes such as Sanger Institute (Cambridge), EMBL (Heidelberg),
Myograd (Berlin), IBRO (Paris). He has also delivered a number of scientific talks at national
and international platforms. He has more than seven years of teaching experience in
Biochemistry with a special focus on Proteomics and Protein Biology, Enzymology, Metabolism,
Radical Biology and Lab techniques.
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Chapter 1: Atomic Structure and Chemical Bonding��������������������������������������������������������������������������3–22

chapter 2: Mole Concept and Concentration Terms������������������������������������������������������������������������23–36

chapter 3: Concept of pH and Biological Buffers�����������������������������������������������������������������������������37–59

Chapter 4: Bioenergetics and Energy Coupling������������������������������������������������������������������������������� 60–82

Chapter 5: Chemical Kinetics & Colligative Properties��������������������������������������������������������������������83–92

Section 2: Biomolecules: Structure and Function

Chapter 6: Amino acids: Structure and Properties������������������������������������������������������������������������� 95–112

Chapter 7: Proteins and their conformations���������������������������������������������������������������������������������113–133

Chapter 8: Proteins: Classification & model examples���������������������������������������������������������������� 134–146

Chapter 9: Carbohydrates: Structure and Functions���������������������������������������������������������������������147–172

Chapter 10: Lipids: Structure and Biological Functions���������������������������������������������������������������� 173–194

Chapter 11: Nucleic Acids: Structural Biochemistry��������������������������������������������������������������������� 195–218

Chapter 12: Stabilizing-interactions in Biomolecules�������������������������������������������������������������������219–236

Section 3: Metabolism and its Regulation

Chapter 15: Enzymes I: Principles of Catalysis�����������������������������������������������������������������������������296–316

Appendix 1: List of Common Biochemical Tests��������������������������������������������������������������������������� 353–356

appendix 4: Credits and Suggested Readings������������������������������������������������������������������������������362–366

chapter 3: Concept of pH and Biological Buffers 47

The above expressions are used to calculate pH of a buffer when the

Further during the entire text some tricks to remember

the difficult topics have also been added, which

Log Salt/Acid will be 0 (Log 1 5 0).

3.4.5 effect of dilution on buffers.

Some practice problems on HH equation

High Yielding Facts

high yielding facts

unique small points about the topic that may

Chapter 1: Atomic Structure and Chemical Bonding 21

QuEStioNS froM PrEViouS ExAMS

Q3: If van der Waals interaction is described by the following relation,

difficult topics are aided with online support. Readers may

Chapter 2: Mole Concept and Concentration Terms 23–36

Chapter 3: Concept of pH and Biological Buffers 37–59

Chapter 4: Bioenergetics and Energy Coupling 60–82

Chapter 5: Chemical Kinetics & Colligative Properties 83–92

1.1.1. Plum Pudding Model

2.3 Common concentration terms: How to make a choice?

Equillibrium Acheived in the Equillibrium Acheived Equillibrium Acheived

Keq < 1 Keq = 1 Keq > 1

Only Reactants Reactants equal Only Products

3.2.2. Acid Dissociation constant

3.2.3 Base Dissociation constant

3.2.4. Representing multiple equilibria

3.2.5. p- Function for equilibrium constants

Compensated Uncompensated Elevated Anion Gap Normal Anion Gap*

Description of alteration in the pH

Chapter 1: Atomic Structure and Chemical Bonding��3–22

chapter 2: Mole Concept and Concentration Terms��23–36

chapter 3: Concept of pH and Biological Buffers��37–59

Chapter 4: Bioenergetics and Energy Coupling�� 60–82

Chapter 5: Chemical Kinetics & Colligative Properties��83–92

Chapter 6: Amino acids: Structure and Properties�� 95–112

Chapter 7: Proteins and their conformations��113–133

Chapter 8: Proteins: Classification & model examples�� 134–146

Chapter 9: Carbohydrates: Structure and Functions��147–172

Chapter 10: Lipids: Structure and Biological Functions�� 173–194

Chapter 11: Nucleic Acids: Structural Biochemistry�� 195–218

Chapter 12: Stabilizing-interactions in Biomolecules��219–236

Chapter 15: Enzymes I: Principles of Catalysis��296–316

Appendix 1: List of Common Biochemical Tests�� 353–356

appendix 4: Credits and Suggested Readings��362–366

Chapter 2: Mole Concept and Concentration Terms 23–36

Chapter 3: Concept of pH and Biological Buffers 37–59

Chapter 4: Bioenergetics and Energy Coupling 60–82

Chapter 5: Chemical Kinetics & Colligative Properties 83–92

Chapter 14: Metabolism of Biomolecules 254–295

Chapter 15: Enzymes I: Principles of catalysis296–316

Chapter 16: Enzymes II: Kinetics317–333

Chapter 17: Enzymes III: Regulation 334–352