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About The Authors Dr. Aditya Arya Mr. Arya is PhD in the area of Nano-medicine and Proteomics from Defence Research and Development Organization, Delhi. He has done M.Sc. in Biochemistry from Madurai Kamaraj University (MKU) and qualified CSIR-JRF (SPM), ICMR, DST-INSPIRE and GATE with top national ranks. He has published 15+ papers in top International Journals and has membership of several International Societies in Nanomedicine. He has more than 5 years of teaching experience in Biochemistry with special focus on Proteomics and Protein Biology, Enzymology, Metabolism, Radical Biology and common lab Techniques.

Dr. Mohit Gupta Mr. Mohit has Doctorate Degree in Applied Physiology, M.Sc. from Delhi University and secured 2nd Rank in the University. He has also qualified CSIR-NET/JRF (SPM). He is also the recipient of National Scholarship by HRD Ministry and was felicitated by a former Prime Minister. Teaching expertise in Molecular Cell Biology, Applied Plant Physiology, Cancer Biology, Cytogenetic and Plant Biotechnology.

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Concise Biochemistry

ConCise BioChemistry

Dr. ADityA AryA Dr. mohit guptA

Concise Biochemistry: for CSIR NET and other competitive exams

Authors: Dr. Aditya Arya and Dr. Mohit Gupta

Grassroots Academy, 2580, Hudson line, Kingsway camp New Delhi - 110009

© 2016. All rights reserved with Dr. Aditya Arya.

ALL RIGHTS RESERVED. No part of this work covered by the copyright herein may be reproduced, transmitted, stored, or used in any form or by any means, graphic, electronic, or mechanical, including but not limited to photocopying, recording, scanning, digitizing, taping, Web distribution, information networks, or information storage and retrieval systems, except as permitted under law.

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First Edition 27 April 2016

Preface I am delighted to present this book entitled “Concise Biochemistry” exclusively published for preparation of CSIRNET/ICMR/GATE and other competitive exams including Master of Science. 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 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. 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 and support 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

x  Concise Biochemistry

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: Strutural 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

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Table of Contents  xi

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 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

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xii  Concise Biochemistry

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

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How this Book helps in CSIR NET/ICMR/other exams preparation? EXAM INDEX This books has been custom written for assisting students in their preparation for competitive exams. Starting of each chapter is gives a detailed view of each chapter and it weightage in exams. We have also provided an exam weightage icon on the scale of 5 which helps readers to know the impact and importance of particular topic based on previous trends.

ChApter

Atomic Structure and Chemical Bonding

1

Learning OBjeCtiveS

exaM 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 textbooks, yet their fundamental constituents are atoms and their chemical properties are governed by the same rules of chemistry.

INTERESTING FACT

interesting Fact The “God particle” is the nickname of a subatomic particle called the Higgs boson. In layman’s terms, different subatomic particles are responsible for giving matter different properties. One of the most mysterious and important properties is mass. Some particles, like protons and neutrons, have mass. Others, like photons, do not. The Higgs boson or “God particle,” is believed to be the particle which gives mass to matter. The “God particle” nickname grew out of the long, drawnout struggles of physicists to find this elusive piece of the cosmic puzzle. What follows is a very brief and simplified explanation of how the Higgs boson fits into modern physics, and how science is attempting to study it.

In order to understand the stability of biomolecules, their interactions and the mechanism of biochemical reactions, it is essential to revisit the atomic structure and basic atomic/molecular interactions that are essential for the existence of every chemical entity in this universe. In this chapter, we will also focus on biological relevance of atomic structure and their interactions.

Another opening shot in the chapter is the column called interesting fact, which include some of the unique facts that will excite the students giving them some unique information on the topic.

The word atom has originated from the Greek word ‘a-tomio‘ which means uncutable or non-divisible. The details of the atom could be elucidated only after the discovery of sub-atomic particles such as electron (JJ Thomson), proton (Goldstein) and neutrons (Chadwick). Since early 1800, the atomic structure has been extensively revised and elaborated by several classical experiments. Chronologically, initial model was plum pudding model 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.

Result from entropic stabilization of solvent

Hydrophobic van der waals

Weakest Three types Kessom,Debye and London

Hydrogen Bond

A special case of dipole-dipole interaction

Quantum mechanical Model

Defined Energy Orbits of electrons

may be Polar or Non-Polar

Strongest in nature

Bohr’s Model

Rutherford’s Model

Nucleus centrally placed and e- spin around

Defined atom as electron cloud around nucleus

Sharing of one e by many atoms

Metallic Coordinate Covalent

Plum Pudding Model

Nuclei (plum) in the electron ocean (pudding)

Formed by sharing of electron

Ionic

Electrostatic in origin

is of four types

High energy >100 KJ

Strong Together Define atom

There are various models of atom

Neutron

electrically neutral

Proton Electron

Relative organization of atom and chorological development is shown below in the Fig. 1.3

Postively charged

Sub Atomic particles

The seminal work of Heisenberg and Schrödinger with ways of describing the quantized energy levels of atoms explained a much better model of atom. Heisenberg used matrices and Schrödinger developed a wave equation. It was solutions of Schrödinger‘s equation that provided pictures of electrons’ probability densities around the nucleus of an atom.

Sharing of lone pairs

Weak

to form molecules

Chemical Bonds

Atoms

COnCept Map

7 Negatively charged

Chapter 1: Atomic Structure and Chemical Bonding

are made up of

Most Importantly, Last part of each chapter is provided with a small concept map of everything that could summaries the chapter. This would provide a bird’s eye view of chapter and help students to revise faster.

are connected by

concept map

Based on strength bonds are of two types

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Less energy Product c.  Reactant 1

Progress of Reaction Only Reactants Present

Reactants equal to Products

Only 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 acidbase 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-

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Chapter 3: Concept of pH and Biological Buffers  41

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+ + OHWhere 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+  PO43- + 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%]

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42  Concise Biochemistry

Then the ratio of product/reactant would be (0.1 3 0.1)/0.9 5 0.01, which represents the equilibrium constant. This can also be written in the form of following formula Ka =

C a2 1- a

Here Ka is equilibrium constant, C is the concentration of unionised component and a is the degree of dissociation.

3.3 Concept of pH As we have discussed, the importance of H1 ion concentration in biological systems, one needs to know and maintain appropriate H1 concentration for simulating such reaction ex situ. Therefore, it is essential to know the methods to calculate and represent H1 concentration. As explained above the p- function makes the calculation much easier; the H1 ion concentration could also be converted into p- function and may be called as pH (negative log of H1 ions).

Online Support Video Lecture Available: You tube channel: video 3.1: Equilibrium and pH.

History of term pH (optional for reading) The concept of pH was first introduced by Danish chemist Søren Peder Lauritz Sørensen at the Carlsberg Laboratory in 1909 and revised to the modern pH in 1924 to accommodate definitions and measurements in terms of electrochemical cells. In the first papers, the notation had the “H” as a subscript to the lowercase “p”, as so: pH. The exact meaning of the “p” in “pH” is disputed, but according to the Carlsberg Foundation pH stands for “power of hydrogen”. It has also been suggested that the “p” stands for the German Potenz (meaning “power”), others refer to french puissance (also meaning “power”, based on the fact that the Carlsberg Laboratory was French-speaking). Another suggestion is that the “p” stands for the Latin terms pondus hydrogenii, potentia hydrogenii, or potential hydrogen. It is also suggested that Sørensen used the letters “p” and “q” (commonly paired letters in mathematics) simply to label the test solution (p) and the reference solution (q). Current usage in chemistry is that p stands for “decimal co-logarithm of”, as also in the term pKa, used for acid dissociation constants pH 5 – log [H1] 5 log 1/[H1]

3.3.1. How was pH scale was set? Now let us understand how the pH scale was set and why 7 was chosen as the midpoint of that scale. This could be understood from the ionization of water molecule. Water is the only molecule that produces equal number of H1 and OH – ions and therefore one of the variable could be easily replaced by other in equations. So, water was an excellent choice for determining the pH scale. The ionic equilibrium of water can be represented as H2O  OH – 1 H1 éOH- ù éH+ ù ê ûú ëê ûú K eq = ë [H2O] From experimental methods (potentiometric titration) the Keq was found to be 1.8310 –16 Also, let us determine the molarity of water (imagine that 1Kg of water is dissolved in itself i.e. one litre, so we can say that 1000 g is present in 1L of solution or number of moles of water in 1L will be 1000/18 5 55.5) Therefore, molarity of water is equal to 55.5 5 [H2O]

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Bioenergetics and Energy Coupling

chap t er

4

Learning ObjectivesExam Index         •• Revisiting the basic laws of thermodynamics in biological perspective •• Concept of Gibb’s free energy and spontaneity of a reaction •• Major energy equivalents in biological systems •• Introduction to reaction kinetics and colligative properties

4.1 Introduction Thermodynamics is a branch of science that involves study of Heat (Thermos) and its variability (Dynamics) over time and space. Fundamental laws of thermodynamics, which have been established for over a century, explain the process of energy transfer in any physical or chemical process. If we observe the living system as a complex network of biochemical reactions then the progress and direction of these reaction can only be explained with the help of thermodynamic principles as stated in the chemistry texts. Even if we consider multicellular life forms as complex machines, the fundamental laws in physics will be perfectly applicable to life forms. This demonstrates that biological systems are also governed by the same set of basic rules and therefore some predictions can be made about the biological processes by simple thermodynamic calculations. In this chapter we will learn about the thermodynamics in biological systems (or Bioenergetics), basically in light of classical laws of physics and chemistry. We will also discuss similarities and differences between the biological and non-biological systems.

Interesting Fact The average adult human, with a typical weight of 70 Kg, consumes approximately 65 Kg of ATP every day, an amount nearly equal to his/her own weight! Fortunately, we have a highly efficient recycling system for ATP/ADP utilization. The energy released from the food is stored transiently in the form of ATP. Once ATP energy is used, ADP and phosphate are released, our bodies recycle it to ATP through intermediary metabolism so that it may be reused. The typical 70-Kg body contains only 50 g of ATP/ADP. Therefore, each ATP molecule in our bodies must recycle 1300 times a day! Were it not this fact, at current commercial prices of about Rs. 1200/g, our ATP habit would cost more than Rs 7.8 crore per day! And the richest businessman on this globe would find it difficult to sustain him and his employees. In these terms, the ability of biochemistry to sustain the marvelous activity and vigour of organisms deserve our respect and fascination

In biology, we encounter many examples of the relationship between energy and work such as muscle contraction and locomotion, movement of ions across cells or cellular compartments, and electrochemical signalling in living organism. Apparently complex and intricate metabolic pathways are infact reflections of basic thermodynamic principles. The synthesis of biological molecules and cell division are also manifestations of work at the molecular level. The energy that produces all this work in our bodies comes from food and ultimately all energy sources converge at sun- the ultimate source of energy for life. See Figure 4.1.

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G (Final) < G (initial) ΔG = Negative

Spontaneous Initial State

Final State

Gibb’s Free Energy (Available for doing work)

Gibb’s Free Energy (Available for doing work)

Chapter 4: Bioenergetics and Energy Coupling  67

G (Final) > G (initial)

ΔG = Positive : Non- Spontaneous Process

ΔG = Postive

ΔG = Negative : Spontaneous Process

NonSpontaneous

ΔG = Zero : Process achieved equillibrium

Final State

As initial state must have some excess free energy (ability to do work) in comparisons to final state therefore there rules hold true

Initial State Progress of reaction

Progress of reaction

Fig 4.5.  Concept of Gibbs free ener]gy change and its relation with spontaneity

The sign of DG depends on the signs of the changes in enthalpy (DH) and entropy (DS), as well as on the absolute temperature (T, in kelvin). DG changes from positive to negative (or vice versa) where T 5 DH/DS. We can further distinguish four cases within the above rule just by examining the signs of the two terms on the right side of the equation. self-test Q:  Consider the equation DG° 5 DH° – TDS°, which of the following statement is NOT CORRECT? a. When DG° is negative, the reaction is exergonic

[IIT-JAM 2010]

b. When DG° is negative, the reaction can occur spontaneously c. When DS° is negative, the molecular disorder decreases during the reaction d. When DH° is negative, the reaction is endothermic Answer.  d, is NOT CORRECT, as negative DH° represents exothermic reaction.

Conditions of Spontaneity

ΔS is Positive (or TΔS negative) Randomness is increasing

ΔS is Negative (or TΔS positive) Randomness is decreasing

ΔH is Positive (Endergonic)

ΔH is Negative (Exergonic)

ΔH is Positive (Endergonic)

ΔH is Negative (Exergonic)

Spontaneous only at High Temperatures

Always Sontaneous

Never Spontaneous

Spontaneous only at low temperatures

Fig 4.6.  Spontaneity at various conditions of enthalpy and entropy, [It is not required to memorize the table but conclusion may be drawn by considering the sign of all factors, and determining the conditions when they give rise to negative Gibbs free energy]

Online Support Video Lecture Available: You tube channel: video 4.2: Understanding Spontaneity

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68  Concise Biochemistry

4.2.5 Conclusion drawn from Third law of thermodynamics The third law of thermodynamics states that the entropy of a perfect crystal at absolute zero (i.e. zero Kelvin temperature) is exactly equal to zero, which means at that temperature matter behaves perfectly crystalline. At zero kelvin the system must be in a state with the Trick to Remember minimum possible energy and therefore temperature below 0 kelvin cannot There have been several question in exam exist. This statement of the third law holds true if the perfect crystal on calculation of Gibbs free energy and has only one minimum energy state. Entropy is related to the number of spontaneity of reaction, the difficult part is possible microstates, and with only one microstate available at zero kelvin, which formulae to be used and what care the entropy is exactly zero. A more general form of the third law applies must be taken to minimise errors following to systems such as glasses that may have more than one minimum energy table summarises usage of various formulae. state. The third law of thermodynamics is sometimes stated as follows: The entropy of a perfect crystal at absolute zero is exactly equal to zero. At zero kelvin the system must be in a state with the minimum possible energy, and this statement of the third law holds true if the perfect crystal has only one minimum energy state. Entropy is related to the number of possible microstates, and with only one microstate available at zero kelvin, the entropy is exactly zero. A more general form of the third law applies to systems such as glasses that may have more than one minimum energy state. Condition

Formula

Use in Biochemistry

Caution while solving

First law of thermodynamics:

DU 5 w 1 q

Calculations involving physiological processes such as locomotion, fluid ascent in plants etc.

Sign conventions of w and q

Heat capacity

C 5 q/DT

Calculating change in temperature or heat evolved/absorved during a bichemical reaction.

Sign conventions of q, temperature in Kelvin

Change in Enthalpy

DH 5 DU 1 PDV

Calculations involving physiological processes such as energetics of breathing.

Sign conventions

Change in entropy

DS 5 qrev/T

Not needed often in Biochemistrry

Standard Gibbs free energy

DG° 5 DH ° – TDS °

For calculating gibbs free energy under standard conditions. (constant temperature (25°C) and 1 Atm pressure

In biology the reference temp is 37°C, T – in kelvin

Gibbs free energy and equilibrium

DG°5 –2.303 RT log K

Finding out equilibrium constant or concentrations at equilibrium, or spontaneity of reaction towards reaching equilibrium

Unit of G in Joules, value of R 5 8.31, T 5 in Kelvin

Gibbs free energy at nonstandard conditions

DG 5 DG°1 2.303 RT log Q

Finding out Gibbs free energy at nonstandard conditions, realistic biochemical conditions when conc are not 1 molar.

Unit of G in Joules, value of R 5 8.31, T 5 in Kelvin

Gibbs free energy and Standard reduction potential

DG° 5 –nF DE°

Finding out Gibbs free energy or spontaneity of redox reaction in mitochondria, chloroplast.

Most of NAD, FAD, FMN based reactions have n 5 2, F 5 96500 E 5 to be used in volts

Gibbs free energy for ion transport (osmotic)

DG 5 DG°1 2.303 RT log [D]/[O] DG° 5 2.303 RT log [D]/[O]

Detrmination of spontenuity of movement of molecules across plasma membrane movement (purely osmotic i.e. molecules are not charged ) or to calculate concentrations across plasma membrane

Unit of G in Joules, value of R 5 8.31, T 5 in Kelvin

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Chapter 4: Bioenergetics and Energy Coupling  69

Condition

Formula

Use in Biochemistry

Caution while solving

Gibbs free energy for ion transport (osmotic1Donnan)

DG 5 DG°1 2.303 RT log [D]/[O]1 nFψ ψ is membrane potential n 5 charge on ion that is transported

Determination of spontenuity of movement of molecules across plasma membrane movement (osmotic and donnan, i.e. molecules are charged ) or to calculate concentrations across plasma membrane

Unit of G in Joules, value of R 5 8.31, T 5 in Kelvin, n is no. of electrons involved, E in volts. F 5 96500

[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

Standard free energy change ΔGº = -35 KJ/Mol

Free energy of hydrolysis is variable ΔG = ΔGº+ RT ln Q

-4.0

ADP + Pi

Ratio of ATP/ADP drives Q and hence Gibbs free energy

Hence the ease of hydrolysis of ATP is not similar at all sub-cellular locations

NAD+

Midpoint redox potential (V)

ATP + H2O

Energetics of ATP hydrolysis (-0.32 V)

NADH

2e -

0.0

FMN

2eFMNH2 (-0.03 V) Ubiquinone (0.06V) 2e-

FAD FADH2

Succinate (0.03V) Fumarate

Fe3+ Cyt b (0.04 V) Fe2+ e-

Fe3+ Cyt c (0.22V) Fe2+ eFe3+ Cyt a (0.22V) Fe2+

0.4

0.8

e-

Cytochrome oxidase

Fe3+ Cyt a3 (0.39V) Fe2+ 2e-

1O 2 2

(0.82 V)

H2O

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.

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Section

2

Chapter 6: Amino acids: Structure and Properties95–112 Chapter 7: Proteins and their conformations

113–133

Chapter 8: Proteins: Classification & model examples134–146 Chapter 9: Carbohydrates: Structure and Functions147–172 Chapter 10: Lipids: Structure and Biological Functions173–194 Chapter 11: Nucleic Acids: Strutural Biochemistry195–218 Chapter 12: Stabilizing-interactions in Biomolecules

219–236

chapt er

Amino acids: Structure and Properties

6

Learning ObjectivesExam Index         •• Understanding the structure of amino acids •• Classification of amino acids •• How to decide charge on an amino acid, concept of isolelectric point (pI) •• Derivatives of amino acids and their metabolic roles.

6.1 Introduction Proteins are the most abundant biomolecules present in the living systems. Infact, Rubisco (an enzyme present in plants used for fixing carbon) is the most abundant biomolecule in the living world. Amino acids are present in thousands of different forms varying both in structure and function. Enzymes, hormones, muscle fibers, haemoglobin, neurotransmitters, eye lens, antibodies, nails, hairs, horns are some of the diverse functional forms of protein. Amino acids constitute the proteins by condensation and forming peptide bonds with each other, chemically amino acids are organic compounds that contains amino and carboxyl groups on the same carbon. Every protein is made up of tiny constituents called amino acids. Let us now have a look at number of amino acids known by answering following questions. How many amino acids are known till date? Often we say 20 or some say 21 or 22, but actual number of amino acids known is around 900. As most of the amino acids have non-protegenic roles such as acting as metabolites, most of them are not readily discussed in text books. However, the number of amino acids present in protein is far less (nearly 35), and those which are incorporated in proteins at the time of protein synthesis are only 22 (rest of them are modified after protein synthesis is completed). Among these 22, only 20 are encoded using standard codons and therefore called as standard amino acids (selenocystein and pyrolysine are added using stop codons). so they are non-standard amino acids.

Interesting Fact The first few amino acids were discovered in the early 19th century. In 1806, French chemists Louis-Nicolas Vauquelin and Pierre Jean Robiquet isolated a compound in asparagus that was subsequently named asparagine, the first amino acid to be discovered. Cystine was discovered in 1810, although its monomer, cysteine, remained undiscovered until 1884.Glycine and leucine were discovered in 1820. Usage of the term amino acid in the English language is from 1898. Proteins were found to yield amino acids after enzymatic digestion or acid hydrolysis. In 1902, Emil Fischer and Franz Hofmeister proposed that proteins are the result of the formation of bonds between the amino groups of one amino acid with the carboxyl group of another, in a linear structure which Fischer termed peptide.

Just to have a recap: •• 20 standard amino acids – coded by standard genetic codes •• 22 proteinogenic amino acids – incorporated in proteins during its synthesis. •• 35 amino acids or their variants are present in proteins. •• Over 80 amino acids created abiotically in high concentrations

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96  Concise Biochemistry

•• About 900 are produced by natural pathways •• Over 118 engineered amino acids have been placed into protein

6.2 Basic structure of amino acids and associated nomenclature 6.2.1 General formula and Nomenclature Each amino acid contain at least one Amino group and carboxyl group present on same carbon, a side chain may or may not be present, side chain may contain straight or branched alkyl groups, alkyl groups associated with additional carboxyl group, or additional amino group. As per IUPAC system amino acids may be called as amino derivatives of carboxylic acids (x-aminoalkanoic acid.). Conventional nomenclature of amino acids is written as a -amino acids as the first carbon at which functional group is attached is called as alpha amino acids, other amino acids such as b, g, d etc. are also possible where amino group is present at beta, gamma or delta positions. However the numbering in IUPAC system starts from carboxyl group so in beta amino acids amino group would be present on 3rd carbon. Fig 6.1 represents general formula of amino acid and difference between its variants arising from different positions of amino group. H H3N

+

C

COOH

+

H3N

R

H

H

C

C

H

R

COOH

H3N

+

H

H

H

C

C

C

H

H

R

COOH

Fig 6.1.  General structure of a -amino acid, R – side chain –varies depending on the type of amino acids

6.2.2 D and L configuration (absolute configuration of amino acids) D and L represent the stereoisomers of a molecules that arise due to difference in the arrangement of atoms in space. But we must note here that D and L may not necessarily represent optical isomers (dextrorotatory and laevorotatory. Every amino acid (except glycine) can occur in two isomeric forms, because of the possibility of forming two different enantiomers (stereoisomerism) around the central carbon atom. By convention, these are called L- and D- forms, analogous to left-handed and right-handed configurations. One should refer to the structure of alanine for determining the D- or L- configuration of amino acids as alanine is considered as reference molecule. Here position of NH3 group decides (if it’s on left side molecule is having L- configuration and if it is on right side molecule is having D- configuration). However, this is a very crude logic to understand this, there are specific rules based on priority groups and relative position of atoms, as per organic chemistry (students may refer standard organic chemistry books for details). Only L-amino acids are manufactured in cells and incorporated into proteins. Therefore, organism fed exclusively on D-amino acids will not survive. Exceptionally some biological systems have D- amino acids, few examples are given below. •• Depsipeptide of NAM in peptidoglycan of gram positive bacteria (A depsipeptide means peptide formed by alternate arrangement of D and L amino acids) •• Some antibiotics – Tyrosidin, Gramicidin •• Marine snake venom peptide – e.g. countryphan L- alanine

D-alanine

H +

H3N

C CH3

Oxygen

H COOH

HOOC

C

H3N

+

CH3

Nitrogen Hydrogen Carbon

Fig 6.2.  Structure of L and D Alanine (note the relative position of amino group)

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Questions from Previous Exams

110  Concise Biochemistry

Questions from Previous Exams Q1. An amino acid contains no ionizable group in its side chain (R). It is titrated from pH 0 to 14. Which of the following ionizable state is not observed during the entire titration in the pH range 0 - 14? [CSIR DEC 2011]

Solution: In this question option c can never exist as this represents a state where amino group has lost its proton but carboxyl group has not. As per the organic chemistry principles, amino group is basic and therefore it will always loose its proton after carboxylic group, state a will exist during pH5pI, state b will exist in highly acidic conditions, while state d, will exist in highly alkaline conditions Q2. Which statement best describes the pKa of amino groups in proteins?

[CSIR JUNE 2013]

a. pKa of amino group is lower than the pKa of a -amino group b. pKa of a -amino group is lower than the pKa of e -amino group. c. pKa of e - amino group is same as the pKa of a -amino group. d. pKa of a -amino group is higher than the pKa of guanidine side chain of arginine. Solution: e –amino group becomes more basic due to 1I effect of side chain that promotes the attraction of proton and therefore it becomes larger than a -amino group, hence b is correct. Q3. In isoelectric focusing experiments, proteins are separated on the basis of their

[CSIR NET June 2014]

a. relative content of only positively charged residues b. relative content of only charged residues c. relative content of positively and negatively charged residues d. mass to charge ratio. Solution: IEF is based on net charge on a peptide which is decided on the basis of both negative and positively charged residues. Q4. A small fraction of clear cellular lysate was run on an isoelectric focusing gel (IEF) to purify a particular protein, which showed a number of sharp bands corresponding to different pI values. The protein of interest has a pI of 5.2 Therefore the band corresponding to pI 5.2 was cut, eluted with appropriate buffer and subjected to SDSPAGE, which showed 3 distinct bands. Which one of the following inferences CANNOT be drawn from the above observation? [CSIR NET JUNE 2014] a. Several different proteins having same pI may be present at the single band on the IEF gel. b. SDS- PAGE showed 3 distinct bands which may represent molecular mass of different proteins. c. The protein of interest may be composed of 3 subunits. d. As the IFE gel showed a distinct band corresponding to p I 5.2 , which is the p I of the protein of interest the protein is composed of a single subunit.141 Solution: c. The protein of interest may be composed of 3 subunits. Could not be concluded on the basis of SDSPAGE, we must run a native PAGE (non-denaturing, non-reducing gel to find the subunits)

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Chapter 6: Amino acids: Structure and Properties  111

a. Ser - Cys

b. Tyr-Phe

c. Lys- Ala

d. Arg-Lys

Solution: Only Lys and alanine differ most in terms of number of carbon atoms (or infact any total no. of atoms) so c. is correct answer Q6. Which of the following pairs of amino acids have Disteromers? a. Ala, val

b. Thr, Ile

c. Ile, Trp

d. Trp, Phe

[IISc, PhD, 2013]

Solution: Only Thr and Ile have two asymmetric carbon atoms so they will have 4 stereoisomers and therefore they can have Disteromers.

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Questions from Previous Exams

Q5. Using site directed mutagenesis four mutants are generated, which pair of mutants have the largest difference in the number of atoms. [GATE-BT 2014]

High Yielding Facts

112  Concise Biochemistry

High Yielding Facts ™™ Gramicidin S, (a type of antibiotic) is a cyclic peptide made of 10 amino acids, including two D- amino acids (D-phenylalanine and D-ornithine), the sequence of peptide is –F-P-V/L-F-P-V/L ™™ Isoleucine is the most hydrophobic and Arginine is the most hydrophillic amino acid ™™ Glycine is the smallest and tryptophan is the largest (heaviest) amino acid. ™™ Opioid peptides from skin of Phyllomedusa species, named Dermorphins and Deltorphins also contain Damino acids. ™™ Recent data show that in the biosynthesis of actinomycin D, the second residue of a dipeptidyl-thioester is converted from the L- to the D-isomer. ™™ The phenolic hydroxyl of tyrosine is significantly more acidic than are the aliphatic hydroxyls of either serine or threonine, having a pKa of about 9.8 in polypeptides. ™™ Tyrosines that are on the surface of a protein will generally have a lower pKa than those that are buried within a protein; ionization yielding the phenolate anion would be exceedingly unstable in the hydrophobic interior of a protein. ™™ The food industry is also a major consumer of amino acids, in particular, glutamic acid, which is used as a flavour enhancer, and Aspartame (aspartyl-phenylalanine-1-methyl ester) as a low-calorie artificial sweetener. ™™ Nullomers are codons that in theory code for an amino acid, however in nature there is a selective bias against using this codon in favour of another, for example bacteria prefer to use CGA instead of AGA to code for arginine. ™™ Certain amino acids can be converted to intermediates of the TCA cycle. Carbons from four groups of amino acids form the TCA cycle intermediates a -ketoglutarate, succinyl CoA, fumarate, and oxaloacetate. Amino acids that form a -ketoglutarate are glutamate, glutamine, proline, arginine, and histidine. ™™ Histidine is often converted to formiminoglutamate (FIGLU). The formimino group is transferred to tetrahydrofolate, and the remaining five carbons form glutamate. Glutamate can be deaminated by glutamate dehydrogenase or transaminated to form a -ketoglutarate. ™™ The conjugate acid (protonated form) of the imidazole side chain in histidine has a pKa of approximately 6.0. This means, at physiologically relevant pH values, relatively small shifts in pH will change its average charge. Below a pH of 6, the imidazole ring is mostly protonated. This makes histidine, one of the most suited amino acid for active site of enzymes.

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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 Regulation

334–352

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chap t er

Global View of Metabolism

13

Learning ObjectivesExam Index     •• Understanding the basics of metabolism •• Metabolic pathways are compartmentalised •• Organ specific metabolism •• How to study metabolism: tracing pathways

13.1 Introduction The term metabolism is derived from the Greek word “Metabolismos” for “change”, or “overthrow”. The history of the scientific study of metabolism spans several centuries and has moved from examining whole animals in early studies, to examining individual metabolic reactions in modern biochemistry. Although the metabolism of living organism is presented by some of the most complex and intermingled networks but for the ease of understanding metabolic pathway may be grouped into three main categories: a. Anabolic pathways are involved in the synthesis of larger and more complex compounds from smaller precursors—e.g. the synthesis of protein from amino acids and the synthesis of reserves of triacylglycerol and glycogen. Anabolic pathways are endothermic. b. Catabolic pathways are involved in the breakdown of larger molecules, commonly involving oxidative reactions; they are usually exothermic and produce reducing equivalents, which are processed mainly via the respiratory chain resulting in the production of ATP.

Interesting Fact The first controlled experiments in human metabolism were published by Santorio Santorio in 1614 in his book Ars de statica medicina. He described how he weighed himself before and after eating, sleep, working, sex, fasting, drinking, and excreting. He found that most of the food he took in was lost through what he called “insensible perspiration” In the 19th century when studying the fermentation of sugar to alcohol by yeast, Louis Pasteur concluded that fermentation was catalyzed by substances within the yeast cells he called “ferments”. He wrote that “alcoholic fermentation is an act correlated with the life and organization of the yeast cells, not with the death or putrefaction of the cells.”

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 8x 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%).

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chap t er

Metabolism of Biomolecules

14

Learning ObjectivesExam Index    •• Understanding the major metabolic pathways of key biomolecules •• Metabolic energetics of carbohydrate and lipid metabolism •• Outline of amino acids and nucleotides metabolism •• Summary of regulation of metabolic pathways

14.1 Metabolism of Carbohydrates The overall metabolism of carbohydrates is a complex process which includes several interconnected biochemical pathways. Rather than looking at individual pathways as discrete biochemical entity, we must look at integrated version of the metabolic pathways which are connected through common metabolic nodes and their flux inside the cell is maintained at a constant level. This gives us better understanding and enables us to understand the regulation of these pathways. Carbohydrates are integral components of diet and serves as primary energy source. Bread, rice, potato, millets and sugar are rich sources of carbohydrates in our diet. The composition of carbohydrates in various food is diverse yet starch and sucrose is the predominant form. Additionally, disaccharides such as maltose and lactose are also present in fruits and milk respectively. In order to understand the metabolism more clearly let us understand it in a stepwise manner. We may begin with a brief note on digestion and absorption, then we will have a glance of bird’s eye view of carbohydrates and integration of all metabolic pathways of carbohydrates. Finally we will note steps and energetics of major pathways and their regulation at local and global levels.

Interesting Fact The metabolome refers to the complete set of small-molecule chemicals found within a biological sample. The biological sample can be a cell, a cellular organelle, an organ, a tissue, a tissue extract, a biofluid or an entire organism. The Human Metabolome Database is a freely available, open-access database containing detailed data on more than 40,000 metabolites that have already been identified or are likely to be found in the human body. These efforts have involved both experimental metabolomic analysis (involving NMR, GC-MS, ICP-MS, LC-MS and HPLC assays) as well as extensive literature mining. According to their data, the human serum metabolome contains at least 4200 different compounds

14.1.1. Digestion and Absorption of carbohydrates The primary function of the carbohydrates in our body is to provide instantaneous energy. Some of the tissues of our body such as brain, rapidly contracting muscles, kidney medulla and some cells such as erythrocytes and sperm cells reply exclusively on glucose (with some exceptions during adverse conditions). However, a major portion of glucose consumption is contributed by muscles which consume nearly 50% of the total glucose intake. Fig. 14.1 depicts the fate/distribution of glucose to several tissues.

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Chapter 14: Metabolism of Biomolecules  255

16.7 g

Brain

18.9 g

8.9 g Glucose in meals (Nearly 100 g)

Liver

Kidney

25.5 g

2.2 g 27.8 g Adipose tissue

Muscle (Glucose) Muscle (Glycogen)

Fig 14.1.  Metabolic fate and distribution of glucose to various tissues and organs

Carbohydrates is also a major component of our diet. Soon after ingestion it is digested with the help of various enzymes. Digestion begins in mouth by enzyme salivary amylase (previously known as Ptylin) that digests starch into limit dextrins. On reaching stomach uncatalyzed acid hydrolysis causes slow hydrolysis of polysachharides. Later pancreatic amylases and intestinal enzymes (dextrinases, lactates, maltase, and sucrase) complete the digestion of carbohydrates in the stomach finally result in the formation of monosaccharides. Most prominent monosaccharides produced after complete digestion of starch are Glucose, Fructose and Galactose. Now these monosaccharides are absorbed through the specific transporters present in enterocytes of intestinal villi (finger like projections). As most of the monosaccharides are hydrophobic in nature, their direct absorption is difficult, specific transporters such as Glucose transporter (a type of uniport protein) plays a dominant role. GLUT-5 selectively transports fructose into the cytosol of enterocyte. Additionally, sodium-glucose symporters (SGLT) also transport glucose into the cells. There are several other type of glucose transporters that help in transport of glucose across the plasma membrane of various tissues, some of them have been described in Table 14.1. The essence of transport by the sodium-dependent hexose transporter involves a series of conformational changes induced by binding and release of sodium and glucose, and can be summarized as follows: 1. The transporter is initially oriented facing into the lumen - at this point it is capable of binding sodium, but not glucose. 2. Sodium binds, inducing a conformational change that opens the glucose-binding pocket. 3. Glucose binds and the transporter reorients in the membrane such that the pockets holding sodium and glucose are moved inside the cell. 4. Sodium dissociates into the cytoplasm, causing glucose binding to destabilize. 5. Glucose dissociates into the cytoplasm and the unloaded transporter reorients back to its original, outward-facing position. Fructose is not co-transported with sodium. Rather it enters the enterocyte by another hexose transporter (GLUT5). Glucose, galactose and fructose are transported out of the enterocyte through another hexose transporter (called GLUT-2) in the basolateral membrane. These monosaccharides then diffuse “down” a concentration gradient into capillary blood within the villus. The figure shown below depicts absorption of carbohydrates into intestinal lumen and trans-cytosis to blood capillaries.

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256  Concise Biochemistry

Table 14.1.  Summary of major types of glucose transporters Metabolic Actions of Insulin and Glucagon Major Km Major sites of expression Protein isoform (mM) Facilitative glucose transporters(GLUT) GlUT1

492

Proposed function

3-7

Ubiquitous distribution in tissue and culture cells

Basal glucose uptake; transport across blood tissue barriers

GLUT2

524

17

Liver, islets, kidney, small intestine

High-capacity low-affinity transport

GLUT3

496

1.4

Brain and nerves cells

Neuronal transport

GLUT4

509

6.6

Muscles , fat, heart

Insulin-regulated transport in muscle and fat

GLUT5

501

Intestine , kidney, testis

Transport of fructose

GLUT6

507

NA

Spleen ,leukocytes, brain

GLUT7

524

0.3

Small intestine, colon, testies

Transport of fructose

GLUT8

477

2

Testis, blastocyst, brain, muscle, adipocytes

Fuel supply of mature spermatozoa

GLUT9

511/540

NA

Liver, kidney

GLUT10

541

0.3

Liver, pancreas

GLUT11

496

NA

Heart, muscle

GLUT12

617

NA

Heart, prostate, mammary gland

HMIT

618/629

NA

Brain H+ / myo-inositol co-transporter

Muscle-specific; fructose transporter

Na+ /glucose cotransporters(SGLT) SGLT1

664

0.2

Kidney, intestine

Glucose reabsorption in intestine and kidney

SGLT2

672

10

Kidney

Low affinity and high selectivity for glucose

SGLT3

660

2

Small intestine, skeleton muscle

Glucose activated Na+ channel

Redrawn after Zhao and Keeting et al., 2007., NA: Not Known.

14.1.2. Metabolic fate of carbohydrates: various catabolic and anabolic pathways Carbohydrates have primary metabolic fate as oxidation into carbon dioxide and water with a concomitant generation of ATPs. This energy generating process is catabolic in nature and achieved by set of three closely related and connected pathways named Glycolysis, citric acid cycle and electron transport chain. An additional catabolic process that occurs primarily in animals include the metabolic breakdown of stored glycogen to generated glycolytic intermediate culminating in energy generation (Glycogenolysis). In contrast to catabolic pathways of carbohydrates, synthesis of new glucose from non-carbohydrate precursors is known as (Gluconeogenesis) and polymerization reaction leading to the formation of glycogen is called Glycogenesis also operate in tissue specific manner. However the catabolic and anabolic pathways usually remain segregated in cellular compartments. Another anabolic pathway called Pentose Phosphate pathway (PPP) operate in cell to generate pentose, hexose, heptose, triose and tetrose from glucose. This pathway is particularly important for the nucleotide formation. In the following sections we will understand each of the metabolic pathways with specific focus on key steps of the pathway, energetics and regulation of the pathway at key steps. The main focus of discussion here is to understand the process and regulation rather than structures of metabolites. (Some of the standard biochemistry text such as Biochemistry by Voet and Voet or Nelson and Cox Lehninger Biochemistry may be referred for details)

A. Glycolysis Glycolysis is the most critical phase in glucose metabolism during cellular respiration. The term “glycolysis” literally means breakdown of glucose and sugars. Biochemically, it involves the breakdown of glucose to pyruvate (or pyruvic acid) via a series of 10 enzymatic reactions occurring in cytoplasm. This pathway is also called as Embden-Meyerhof-Paranas Pathway on the name of its discoverers. Glycolysis is said to occur in two phases first preparatory phase in which Glucose is converted to Glyceradehyde3 phosphate after 4 reactions. The second phase is called Pay-off Phase in which reducing equivalents and ATP is generated from the conversion of Glyceraldehyde-3-Phosphate (GADP) to the final product pyruvate after 6 reactions. Complete pathway with description of enzyme and energy generation steps is shown in Fig 14.3. © 2016. Aditya Arya, Grassroots Academy. All Rights reserved. Unauthorised Photocopying and distribution will be treated under Law

Chapter 14: Metabolism of Biomolecules  257

CARBOHYDRATE METABOLISM Cellulose

Starch

Cellulase (NOT in Humans)

Digestion in stomach Enzyme : none Acid hydrolysis by HCl

Sucrose

Sucrase

Maltase

2

Amylase

Digestion in mouth (20-40%) Enzyme : Salivary amylase

Lactase

1

Maltose Lactose

Glucose

3

Glucose Fructose Galactose Glucose Glucose

Digestion in duodenums (50-80%) Glucose Enzyme: amylase

4

Digestion in illeum (~100%) Enzyme maltase, lactase, sucrase, dextrinase

5

Absorption in illeum through villi, via specific tranporters into the adjoining capillaries

Villi

serosa

Jejunal mucosal cell

Lumen

Fructose villi- capillaries

Glucose/Galactose

Glucose Glycolysis

Gluconeogenesis

Pyruvate

Na

+

K

+

+

Na+ Na+/K+ Pump

All monosaccharides are transported to liver via hepatic portal system followed by metabolic conversion or transport to various organs

ATP

Glycogenolysis

Glycogen Glycogenesis

Ribose Pentose Phpsphate Pathway

7

Various metabolic pathways at sub-cellular level and generation of energy (ATP) and other useful metabolites

Citric Acid Cycle

Acetyl coA

Fermentation

Lactate

GLUT2

SGLT1

Na

6

Pentoses GLUT5

Capillary in villi

Pentoses

Fatty acid biosynthesis

H2O

Electron Transport Chain

CO2

ATPs

NADH

Fatty Acids

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Fig. 14.2  Overview of carbohydrate metabolism in human

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258  Concise Biochemistry

Pathway Glucose Hexokinase Glucose-6-Phosphate

Preparatory Phase

ATP ADP

Energetics

Regulation

ATP per Glucose molecule

Insulin Glucagon

Aerobic conditions (Heart, Liver, Kidney)

Phosphoexose isomerase Fructose-6-Phosphate

ATP ADP

2,6-Bisphosphoglycerate; AMP

Phosphofructokinase

ATP, Citrate

Fructose-1,6-biphosphate

Aldolase Phosphotriose isomerase Glyceraldehyde-3-Phosphate Dihydroxyacetone phosphate

NAD+ H+ NADH

3-Phosphoglyceraldehyde Dehydrogenase 1,3- Bisphosphoglycerate

ADP

Payoff Phase

ATP

Phosphoglycerate kinase

ATP per Glucose molecule Aerobic conditions (Brain, Skeletal Muscle) Substrate level phosphorylation : + 4ATPs From NADH (via Glycerate shuttle) : + 3ATPs ATP cosumption : - 2ATPs ___________________________________ NET ATP gain 5 ATPs

3- Phosphoglycerate ATP per Glucose molecule

Phosphoglycerate mutase

Anaerobic conditions

2- Phosphoglycerate Enolase Phosphoenolpyruvate ADP ATP

Substrate level phosphorylation : +4 ATPs From NADH (via malate shuttle) : +5 ATPs ATP cosumption : -2ATPs ___________________________________ NET ATP gain 7 ATPs

Pyruvate kinase Pyruvate

Fructose 1,6-bisphosphate; AMP ATP; Acetyl CoA; Alanine, Glucagon Activation

Substrate level phosphorylation : + 4 ATPs NADH is recycled to NAD+ : + 0 ATPs ATP cosumption : + 2 ATPs ___________________________________ NET ATP gain 2 ATPs

Inhibition

Fig 14.3 Overview of Glycolysis, its regulation and energy fate, Note that glycolysis is regulated at three critical checkpoints highlighted (in yellow). These reactions have large Keq values (irreversible) and the enzymes are mostly regulated by tight allosteric regulation and by covalent modification

It may be interesting to know why glucose is converted to glucose to glucose 6 phosphate in first step of glycolysis. The primary reason is thermodynamic consideration which elevates the energy state of molecule and the second reason is low permeability of Glucose-6-phophate across membranes, so glucose is confined within cytosol. Further, the conversion isomerization of glucose 6-phosphate to fructose-6-phosphate is important for the second phosphorylation which occurs at C1 (an aldehyde group is less susceptible to phosphorylation). Although the Glucose-6-phosphate formed during glycolysis may divert to the formation of Glycogen, heteropolysachharides or feed pentose phosphate pathway. The commitment of Glucose to glycolysis occurs at third reaction catalysed by Phosphofructokinase and therefor this enzyme is most tightly regulated enzyme of glycolysis.

Trick to Remember Every biochemical pathway begins with the activation of low energy molecule into a high energy molecule. This favours the downward reactions on the basis of Gibbs free energy concept. It’s like filling water in dam and increasing its potential energy. The water on falling from the dam loses that energy and generates electricity, same is true with energy generating pathways.

What happens to end products of Glycolysis? The metabolic fate of Pyruvate varies depending on the surrounding conditions. In abundance of oxygen (aerobic conditions) pyruvate is converted to acetyl CoA. While in absence of oxygen (anaerobic conditions) Pyruvate is converted to Lactate to regenerate NAD1 which further promotes ATP generation by speeding up the glycolysis (otherwise glycolysis will stop due to accumulation of NADH). Moreover, in microorganisms the pyruvate is anaerobically converted in ethanol by a process called fermentation.

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chap t er

Enzymes III Regulation

17

Learning ObjectivesExam Index       •• Understanding why regulation is needed •• Basic classification of regulatory mechanisms •• Effect of various enzyme regulations on enzyme kinetics •• Understanding the role of inhibitors in developing drugs and studying biology

17.1. Introduction

Interesting Fact As we learnt in previous chapters that enzymes are pivotal to biochemical reactions, their roles in driving the metabolism is indispensable. However, Arslan et al. introduced intramolecular crosslinks a cell has enormously complicated network of pathways and therefore it between two domains of the Escherichia coli is in order to keep the metabolic flux balanced, the enzymes which are helicase Rep, which unwinds DNA. By inserting catalysing those reaction must be regulated. (Otherwise condition would be linkers of different lengths, the domains can like driverless cars running without control causing chaos). This chapter on be held either “open” or “closed.” The closed enzymology provides a comprehensive information on regulation of enzymes. conformation activates the helicase, but it Understanding regulation of enzyme has become clinically important domain can also generate super-helicases capable of of science, as most of the enzymes are prime targets for developing drugs unzipping long stretches of DNA at high speed and with considerable force. Comstock et al. used which either inhibit or augment the enzymatic activities. It is therefore very optical tweezers and fluorescence microscopy to important to focus on fundamentals of enzyme regulation and to understand simultaneously measure the structure and function alterations in their kinetics during such regulation. The cell has several of the bacterial helicase UvrD. They monitored its mechanisms to regulate enzyme activity, first and the generalised mode of DNA winding and unwinding activity and its shape regulation is compartmentalization of metabolism (discussed in Chapter 13), during these activities. The motor domain also has usually the anabolic and catabolic compartments are separate and enzymes a “closed” conformation during DNA unwinding and have isoforms with different kinetics abilities in various compartments. The switches to a reversed “open” conformation during the zipping-up interaction (Sciecne, 2015 (348) second mode of regulation of enzyme activity is availability of accessory factors (co-factors) for the enzymes, which are either obtained exogenously (vitamins) or produced endogenously. The Third mode of regulation is via regulation of substrate concentration, if there is no substrate, no product will be formed. However, the above modes do show enzyme specificity and therefore operates at global levels.. There are some specific routes of enzyme regulation which specifically modulate enzyme thereby affecting its activity. In the following section we will consider such specific regulatory mechanisms. Trick to Remember Regulation of enzymes is like controlling electrical devices in household or industry. For safety purpose there are some general 17.2. Types of Enzyme regulation

cut-outs (like MCBs) and centralised switches which can switch off the whole power connection of a building (analogous to

Although there are several ways to classify the regulatory mechanisms of we enzymes but basedswitches on their(analogous kinetics studies and the global regulation), while for specific regulation of an electrical appliance have individual to specific basic distinction in mechanism of regulation we may organize enzyme regulation into four major classes. 1. Allosteric regulation, regulation). Sometimes those devices which are frequently used and need to be operated from multiple places may have 2. Regulation by covalent 3. Regulation peptidal 4. (on Regulation by or selective inhibitor. multiple switches like amodification, bulb on staircase may havebymore thancleavage, one switch each floor) room light may have additional switch close to bed, similar to those enzymes which are regulated (analogous to allosteric regulation) .

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336  Concise Biochemistry

In general appearance, the active site is usually a groove or pocket of the enzyme which can be located in a deep tunnel within the enzyme or between the interfaces of multimeric enzymes. There are two proposed models of how enzymes fit to their specific substrate: the lock and key model and the induced fit model (Fig 17.2). Key and lock hypothesis: Emil Fischer proposed the lock and key model, this assumes that the active site is a perfect fit for a specific substrate and that once the substrate binds to the enzyme no further modification occurs. However, this hypothesis could not explain the extra-stability of transition state. Induced fit hypothesis: This was a modified version of key and lock hypothesis given by Daniel Koshland. The induced fit model is a development of the lock-and-key model and assumes that an active site is flexible and it changes shape until the substrate is completely bound. The substrate is thought to induce a change in the shape of the active site. The hypothesis also predicts that the presence of certain residues (amino acids) in the active site will encourage the enzyme to locate the correct substrate. Conformational changes may then occur as the substrate is bound. Lock and Key Model (Emil Fischer) Enzyme

Induced Fit Model (Koshland) Enzyme

ES complex

Substrate

ES complex

Substrate

Transition state structure © 2016. Aditya Arya, all rights reserved

Fig. 17.2.  Difference between Key and Lock hypothesis and induced fit hypothesis

17.3.2. Key features of Allosteric enzymes Historically, the primary studies on allosteric enzymes were performed on aspartate transcarbamylase (ATCase). Aspartate carbamoyltransferase (or aspartate transcarbamylase, ATCase) plays a central role in the regulation of the pyrimidine pathway in bacteria. The Holoenzyme is a dodecamer composed of six catalytic chains, each with an active site, and six regulatory chains lacking catalytic activity. The catalytic subunits exist as a dimer of catalytic trimers, (2xC3), while the regulatory subunits exist as a trimer of regulatory dimers, (3xR2), therefore the complete holoenzyme can be represented as (C3)2(R2) 3. The association of the catalytic subunits C3 with the regulatory subunits R2 is responsible for the establishment of positive cooperativity between catalytic sites for the binding of aspartate and it dictates the pattern of allosteric response toward nucleotide effectors. Fig 17.3 illustrates basic strategy to demonstrate the allosteric nature of the enzyme. NATIVE SDS+BME

Native protein

Add substrate and accessory factors Isolated and Purified

catalyzed the reaction (Sigmoidal Kinetics) Affected by adding CTP and ATP

catalyzed the reaction Kinetics (Michaelis Menten type) NO effect of adding CTP or ATP

Isolated and Purified Electrophoresis

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NOT catalysed the reaction but binds to modulators

C3

C3

Catalytic Unit (Dimer of Trimers) R2

R2

R2 Regulatory Unit (Trimer of Dimers)

Fig. 17.3.  Basic experiment to illustrate the salient features of allosteric enzymes using ATCase as model

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AP P E ND IX

List of Common Biochemical Tests

Test

1

Detection

Reagent

Test results

Comments

All alpha amino acids

Ninhydrin

Purple color (at pH 4-8)

Proline gives yellow colour

Tests for Amino Acids Ninhydrin test

Mechanism: (triketohydrindene hydrate), terminal amines of lysine residues react with ninhydrin Xanthoproteic test

Only Aromatic amino acids

Conc. Nitric Acid

Yellow colour

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). Millon’s test

Phenolic amino acids (Tyrosine)

Mercuric sulphate in sulphuric acid.

Red Colour

Mechanism: he red colour is probably due to a mercury salt of nitrated tyrosine. Histidine test

Bromination of histidine in acid solution

Bromine water

Blue/Violet

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. Hopkins cole test

Only for tryptophan

glyoxilic acid 1 sulphuric acid

Purple

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 cmpond)

a - naphthol

Red Colour

Under alkaline condition

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. Lead sulphide test

boiling with sodium hydroxide (hot alkali)

lead acetate

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. Folin’s McCarthy Test

Imino acids such as Proline and HyPro

isatin reagent

blue colour

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 test

Proline and hydroxyproline

isatin reagent under alkaline condition

Blue Colour

Also detects methionine.

Mechanism: Imino acids such as Proline and hydroxyproline condense with isatin reagent under alkaline condition to yield blue colored adduct.

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AP P E ND IX

2

List of Common Inhibitors

CYTOSKELETAL & CELL DIVISION INHIBITORS Sr. No.

Inhibitor

Mode of Action

Additional comments

1.

CYTOCHALASIN- D

Binds plus end of Actin filament and Prevent Elongation

2.

LATRUNCULIN

Binds G Actin monomers and prevent them from polymerizing into filament.

3.

PHALLOIDIN

Binds tightly all along the sides of actin filament and stabilizes them against depolymerization.

4.

JASPLKINOLIDE

Induces Actin polymerization and stabilizes F actin.

5.

SWINHOLIDE

Severs filament

6.

THYMOSIN

Actin monomer binding protein in mammalian cells.

Naturally occurs in animal cells to regulate cytoskeketan

7.

COLCHICINE

Binds to Tubulin subunit and prevent their polymerization

Obtained from the bark of Chinchona, inhibit spindle formation

8.

NOCODAZOLE

Causes microtubule to depolarize to Tubulin subunit

9.

COLCEMID

Binds to Tubulin subunit and prevent their polymerization

10.

TAXOL

Binds and stabilize microtubules, preventing their depolymerization

11.

VINBLASTINE

Binds to beta Tubulin to prevent polymerization

12.

VINCRISTINE

Binds free Tubulin to prevent Polymerization

All of these inhibitors can prevent contractile ring formation at the time of cytokinesis as Actin is a major component of contractile ring in animal cells

Obtained from Yew tree, inhibit retraction of spindles

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

2.

DIGITOXIGENIN

Digitalis purpura

Inhibit Na-K pump by binding on K binding site

3.

PALYTOXIN

Coral

Inhibit Na-K pump

Used as cardiotonic steroid as they also inhibit Na-K pump to increase which in turn block Na-Ca antiport and elevate Ca in heart and improve efficiency.

4.

TETRODOTOXIN

Puffer fish

Block Na channel

5.

SAXITOXIN

Gonyaulax

Inhibit voltage gated Na Channel

6.

VERATRIDINE

Sabadilla

Bind to Na channel and leave it permanently open

Puffer fish is also called as swell fish or blow fish

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