(BQ) Part 1 book “Enzymes - Biochemistry, biotechnology and clinical chemistry” has contents: An introduction to enzymes, the structure of proteins, the biosynthesis and properties of proteins, specificity of enzyme action, monomeric and oligomeric enzymes, an introduction to bioenergetics, catalysis and kinetics,… and other contents.
ENZYMES: Biochemistry, Biotechnology and Clinical Chemistry Second Edition "Talking of education, people have now a-days" (said he) "got a strange opinion that every thing should be taught by lectures Now, I cannot see that lectures can so much good as reading the books from which the lectures are taken I know nothing that can be best taught by lectures, except where experiments are to be shewn You may teach chymestry by lectures - You might teach making of shoes by lectures!" James Boswell: Life ofSamuel Johnson, 1766 ABOUT THE AUTHORS Trevor Palmer was born in South Yorkshire and graduated from Cambridge University in 1966 with an honours degree in biochemistry, being influenced by (amongst others) Peter Sykes in organic chemistry and Malcolm Dixon in enzymology He then worked as a clinical biochemist at the Queen Elizabeth Hospital for Children, linked to the Institute of Child Health, University of London, obtaining a PhD for research into inherited disorders From this emerged the two main interests of his subsequent career, enzymology and evolution, the latter stimulating a further interest in the long-term effects of natural catastrophes He moved to Nottingham Trent University (then Trent Polytechnic) in 1974, initially as a lecturer in biochemistry, before becoming Head of Department of Life Sciences (1987), Dean of the Faculty of Science and Mathematics (1992), Senior Dean of the University (1998) and Pro Vice-Chancellor for Academic Development (2002), returning to predominantly academic activity as Emeritus Professor in 2006 His books include Understanding Enzymes (1981), Principles of Enzymology for Technological Applications (1993), Controversy - Catastrophism and Evolution (1999) and Perilous Planet Earth (2003) His wife, Jan, teaches psychology and sociology (and is currently a part-time PhD student at Leicester University) Their son, James, is carrying out postdoctoral studies as a Leverhulme Fellow at Nottingham University and their daughter, Caroline, is researching for a PhD at Sheffield University Philip L Bonner went to school in Coventry, West Midlands, before graduating from the University of Sussex in 1978 with an honours degree in biochemistry He then worked as a research assistant at Glaxo plc on Merseyside before leaving to take up a Research Assistant/Demonstrator post at Trent Polytechnic, where he obtained a PhD for research concerning enzymes associated with seed germination Several postdoctoral appointments followed, at Bristol, Lancaster and Central Lancashire Universities, working on a variety of topics including relaxin, aspartate kinase and phospholipase C, before he was appointed as Senior Lecturer at Nottingham Trent University in 1991 There, he has maintained his research interests in enzymology and analytical biochemistry, working on the role of transglutaminase in plant/animal tissue and methods to isolate and characterise post-translationally-modified MHC peptides His first singleauthor book, on protein purification, was published in 2007 His wife, Liz, is a manager of an occupational therapist team in Nottingham and their daughter, Francesca, is at junior school ENZYMES: Biochemistry, Biotechnology and Clinical Chemistry Second Edition Trevor Palmer, BA, PhD, CBiol, FIBiol, FIBMS, FHEA Emeritus Professor in Life Sciences Nottingham Trent University Philip L Bonner, BSc, PhD Senior Lecturer in Biochemistry Nottingham Trent University WP WOODHEAD PUBLISHING ~ ~ Oxford Cambridge Philadelphia New Delhi For: Caroline, Francesca, James, Jan and Liz Published by Woodhead Publishing Limited, 80 High Street, Sawston, Cambridge CB22 3HJ, UK www.woodheadpublishing.com Woodhead Publishing, 1518 Walnut Street, Suite 1100, Philadelphia, PA 19102-3406, USA Woodhead Publishing India Private Limited, G-2, Vardaan House, 7/28 Ansari Road, Daryaganj, New Delhi – 110002, India www.woodheadpublishingindia.com First edition published by Horwood Publishing Limited, 2001 Second edition published by Horwood Publishing Limited, 2007 Reprinted by Woodhead Publishing Limited, 2011 © T Palmer and P.L Bonner, 2007 The authors have asserted their moral rights This book contains information obtained from authentic and highly regarded sources Reprinted material is quoted with permission, and sources are indicated Reasonable efforts have been made to publish reliable data and information, but the authors and the publisher cannot assume responsibility for the validity of all 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is available from the British Library ISBN 978-1-904275-27-5 Printed by Lightning Source Table of Contents Authors' preface xiv Part : Structure and function ofenzymes An introduction to enzymes 1.1 What are enzymes? 1.2 A brief history of enzymes 1.3 The naming and classification of enzymes 1.3 Why classify enzymes? 1.3 The Enzyme Commission's system of classification 1.3.3 The Enzyme Commission's recommendations on nomenclature 1.3.4 The six main classes of enzymes Summary of Chapter 11 Further reading 11 Problems 11 The structure of proteins 2.1 Introduction 14 2.2 Amino acids, the building blocks of proteins 15 2.2.1 Structure and classification of amino acids 15 2.2.2 Stereochemistry of amino acids 17 2.3 The basis of protein structure 18 2.3.1 Levels of protein structure 18 2.3.2 Bonds involved in the maintenance of protein structure 19 2.4 The determination of primary structure 21 2.4.1 The isolation of each polypeptide chain 21 2.4.2 Determination of the amino acid composition of each polypeptide chain 24 2.4.3 Determination of the amino acid sequence of each polypeptide chain 26 2.4.4 Determination of the positions of disulphide bridges 29 2.4.5 Some results of experimental investigation of primary structure .29 2.4.6 Indirect determination of primary structure 30 2.5 The determination of protein structure by X-ray crystallography .30 2.5.1 The principles of X-ray crystallography 30 2.5.2 Some results of X-ray crystallography 35 2.6 The investigation of protein structure in solution .40 vi Table of Contents Summary of Chapter 42 Further reading 42 Problems 43 The biosynthesis and properties of proteins The biosynthesis of proteins 44 3.1.1 The central dogma of molecular genetics .44 3.1.2 The double-helix structure of DNA .46 1.3 The translation of genetic information into protein structure 48 3.1.4 Modification of protein structure after translation 51 3.1.5 Control of protein synthesis .52 3.1.6 Sequence determination 55 3.2 The properties of proteins 57 3.2.1 Chemical properties of proteins 57 3.2.2 Acid-base properties of proteins 58 3.2.3 The solubility of globular proteins 62 Summary of Chapter 64 Further reading 64 Problems 65 Specificity of enzyme action 4.1 Types of specificity 67 4.2 The active site 68 4.3 The Fischer 'lock-and-key' hypothesis 70 4.4 The Koshland 'induced-fit' hypothesis 70 4.5 Hypotheses involving strain or transition-state stabilization 72 4.6 Further comments on specificity 73 Summary of Chapter 74 Further reading 75 Monomeric and oligomeric enzymes 5.1 Monomeric enzymes 76 5.1.1 Introduction 76 5.1.2 The serine proteases 76 5.1.3 Some other monomeric enzymes 78 5.2 Oligomeric enzymes 79 5.2.1 Introduction 79 5.2.2 Lactate dehydrogenase 79 5.2.3 Lactose synthase 81 5.2.4 Tryptophan synthase 81 5.2.5 The pyruvate dehydrogenase multienzyme complex 82 Summary of Chapter 83 Further reading 83 Table of Contents vii Part : Kinetic and chemical mechanisms of enzyme-catalysed reactions An introduction to bioenergetics, catalysis and kinetics 6.1 Some concepts ofbioenergetics 85 6.1.1 The first and second laws of thermodynamics 85 6.1.2 Enthalpy, entropy and free energy 85 6.1.3 Free energy and chemical reactions 86 6.1.4 Standard free energy 87 6.1.5 Bioenergetics and the living cell 88 6.2 Factors affecting the rates of chemical reactions 89 6.2.1 The collision theory 89 6.2.2 Activation energy and the transition-state theory 89 6.2.3 Catalysis 92 6.3 Kinetics ofuncatalysed chemical reactions 93 6.3.l The Law of Mass Action and the order ofreaction 93 6.3.2 The use of initial velocity 95 6.4 Kinetics of enzyme-catalysed reactions: an historical introduction 96 6.5 Methods used for investigating the kinetics of enzyme-catalysed reactions 98 6.5.1 Initial velocity studies 98 6.5.2 Rapid-reaction techniques 100 6.6 The nature of enzyme catalysis 100 Summary of Chapter 102 Further reading 102 Problems 102 Kinetics of single-substrate enzyme-catalysed reactions The relationship between initial velocity and substrate concentration 105 7.1.1 The Henri and Michaelis-Menten equations 105 7.1.2 The Briggs-Haldane modification of the Michaelis-Menten equation 107 7.1.3 The significance of the Michaelis-Menten equation 109 1.4 The Lineweaver-Burk plot 111 7.1.5 The Eadie-Hofstee and Hanes plots 112 7.1.6 The Eisenthal and Comish-Bowden plot 114 7.1.7 The Haldane relationship for reversible reactions 115 Rapid-reaction kinetics 116 7.2.1 Pre-steady-state kinetics 116 7.2.2 Relaxation kinetics 120 7.3 The King and Altman procedure 121 Sunimary of Chapter 124 Further reading 124 Problems 125 viii Table of Contents Enzyme inhibition 8.1 Introduction 126 8.2 Reversible inhibition 126 8.2.1 Competitive inhibition 126 8.2.2 Uncompetitive inhibition 133 8.2.3 Non-competitive inhibition 136 8.2.4 Mixed inhibition 140 8.2.5 Partial inhibition 143 8.2.6 Substrate inhibition 144 8.2.7 Allosteric inhibition 146 8.3 Irreversible inhibition 147 Summary of Chapter 149 Further reading 150 Problems 150 Kinetics of multi-substrate enzyme-catalysed reactions 9.1 Examples of possible mechanisms 153 9.1.1 Introduction 153 1.2 Ping-pong bi-bi mechanism 153 9.1.3 Random-order mechanism 154 9.1.4 Compulsory-order mechanism 154 9.2 Steady-state kinetics 155 9.2.1 The general rate equation of Alberty 155 9.2.2 Plots for mechanisms which follow the general rate equation 157 9.2.3 The general rate equation of Dalziel 158 9.2.4 Rate constants and the constants of Alberty and Dalziel 158 Investigation of reaction mechanisms using steady-state methods 160 9.3.1 The use of primary plots 160 The use of inhibitors which compete with substrates for binding sites 161 9.4 Investigation of reaction mechanisms using non-steady-state methods 165 9.4.1 Isotope exchange at equilibrium 165 9.4.2 Rapid-reaction studies 167 Summary of Chapter 168 Further reading 168 Problems 168 10 The investigation of active site structure 10.1 The identification of binding sites and catalytic sites 173 10.1.l Trapping the enzyme-substrate complex 173 10.1.2 The use of substrate analogues 174 10.1.3 Enzyme modification by chemical procedures affecting amino acid side chains 175 Table of Contents ix 10.1.4 Enzyme modification by treatment with proteases 179 10.1.5 Enzyme modification by site-directed mutagenesis 179 10.1.6 The effect of changing pH 180 10.2 The investigation of the three-dimensional structures of active sites 185 Summary of Chapter 10 187 Further reading 187 Problem 188 11 The chemical nature of enzyme catalysis 11.1 An introduction to reaction mechanisms in organic chemistry 189 11.2 Mechanisms of catalysis 191 11.2.1 Acid-base catalysis 191 11.2.2 Electrostatic catalysis 192 11.2.3 Covalent catalysis 192 11.2.4 Enzyme catalysis 193 11.3 Mechanisms of reactions catalysed by enzymes without cofactors 194 11.3.1 Introduction 194 11.3.2 Chymotrypsin 194 11.3.3 Ribonuclease 195 11.3.4 Lysozyme 196 11.3.5 Triose phosphate isomerase 199 11.4 Metal-activated enzymes and metalloenzymes 200 11.4.1 Introduction 200 11.4.2 Activation by alkali metal cations (Na+ and K+) 200 11.4.3 Activation by alkaline earth metal cations (Ca2+ and Mg2l 201 11.4.4 Activation by transition metal cations (Cu, Zn, Mo, Fe and Co cations) 202 11.5 The involvement of coenzymes in enzyme-catalysed reactions 204 11.5.1 Introduction 204 11.5.2 Nicotinamide nucleotides (NAD+ and NADPl 205 11.5.3 Flavin nucleotides (FMN and FAD) 207 11.5.4 Adenosine phosphates (ATP, ADP and AMP) 210 11.5.5 Coenzyme A (CoA.SH) 211 11.5.6 Thiamine pyrophosphate (TPP) 212 11.5.7 Pyridoxal phosphate 214 11.5.8 Biotin 216 11.5.9 Tetrahydrofolate 217 11.5.10 Coenzyme B 12 218 Summary of Chapter 11 220 Further reading 220 12 The binding of ligands to proteins 12.1 Introduction 222 Sec 11.5] Coenzymes in enzyme-catalysed reactions 207 The zinc ion then acts as an electrostatic catalyst to stabilize the negativelycharged transition-state, while a hydride ion is transferred to NAD+, becoming the HR atom of NADH For the reverse reaction, the zinc ion acts as an electrophilic catalyst to enhance the polarization of the carbonyl group of the substrate and facilitate hydride transfer from the reduced coenzyme As demonstrated by later studies, the catalytic process is facilitated by conformational changes in the enzyme as the reaction proceeds Dogfish muscle lactate dehydrogenase (LDH), which catalyses the interconversion of lactate and pyruvate (see sections 1.3.4 and 5.2.2), is another NAD-utilizing dehydrogenase whose mechanism has been investigated in detail (see section 10.2 and Fig 12.7) In this case, no metal ions are involved LDH is a tetrameric enzyme, each sub-unit having a binding-site for NAD+ As with ADH, the coenzyme binds to part of a "Rossmann fold" The overall reaction has a compulsory-order mechanism, the coenzyme binding first and bringing about a conformational change in the enzyme which enables the substrate to bind close to the nicotinamide ring Substrate-binding, in turn, causes a peptide loop of the enzyme to close over the active site From the studies of Margaret Adams and others in the early 1970s, it was concluded that the reaction mechanism involves: n I H -:N o) substrate(lactate) coenzyme CH3- \ NH o/ IHis-195 (11.27) I H2N:"!- _J C-c(0 + ;i.=-C-NHIArg-171 : :;NAD- r=o glutathione disulphide I -s -G H GSH glutathione S-S-G I CH s-s-a I CH2 Cys-58 Cys-58 I I It can be seen that histidine-467 acts as a base catalyst to assist the splitting of the disulphide bridge of the substrate, and then as an acid catalyst to promote the formation of glutathione, the positive charge carried by the imidazole ring during part of the sequence being stabilized by glutamate-472 The reaction is completed by nucleophilic attack by the sulphur atom of cysteine63 on that of cysteine-58, which allows release of the second molecule of product, leaving the enzyme in its original oxidized form 11.5.4 Adenosine phosphates (ATP, ADP and AMP) The nucleoside phosphates ATP, ADP and AMP are involved in phosphate transfer reactions Sec 11.5] ~ -0 -1o- Coenzymes in enzyme-catalysed reactions ~ ~ -r- -r0 o- o- -q adenine ~ ~ o- 211 - -r_- -r- -~ denine ATP ADP OH OH OH OH -o-P-O-CH2 I o- ~adenine AMP OH OH ATP and ADP may be interconverted by the reaction: ATP + H20 ~ ADP + Pi This tends to go strongly in the forward direction as written (t:J Ge' = -31.0 kJ mor') because the four negative charges which are in close proximity on ATP make it an unstable molecule (although the product, ADP, itself has three negative charges close together), and because the reverse reaction requires negatively-charged ADP to react with negatively-charged Pi The reaction can be coupled to others, so that phosphate may be transferred between ATP and other organic compounds without ever being present as free Pi (section 6.1.5) The importance of ATP in energy metabolism is that, by comparison to other organic phosphates, it is only moderately unstable Hence it may be synthesized by the transfer of phosphate to ADP from a more unstable organic phosphate (e.g phosphoenolpyruvate) by substrate-level phosphorylation, or by oxidative phosphorylation However, ATP is sufficiently unstable to be able to force the transfer of the phosphate to a whole variety of other compounds, thus driving such processes as biosynthesis, active transport and muscular contraction In the cell, adenosine phosphates are stabilized by binding to Mg2+ ions, and their metabolism is strictly mediated by enzymes (see section 11.4.3) In some instances, two phosphate groups, rather than one, may be removed from ATP to liberate inorganic pyrophosphate: ATP + H20 ~ AMP + (PP)i Adenosine phosphates, like the nicotinamide nucleotides, are loosely bound by enzymes and may be regarded both as coenzymes and as co-substrates/co-products of the reactions in which they participate 11.5.5 Coenzyme A (CoA.SH) Coenzyme A has the structure: HS.CH2CH2NH-pantothenic acid-OP0 3-.P03- -ribose-3-P-adenine 212 The Chemical Nature of Enzyme Catalysis [Ch 11 With carboxylic acids it can form thioesters: RC02H + HS.CoA R.COSCoA + H20 ~ (11.35) acyl-CoA These thioesters are of great importance in biochemical metabolism since they can be attacked by electrophiles (including other acyl-CoA molecules and C02) to form addition compounds, and by nucleophiles (including water) to displace the -SCoA group: H 8- (I tW R-c-c-scoA /18f H 8~ (11.36) nucleophile electrophile Some examples are given in sections 11.5.6, 11.5.8 and 11.5.10 11.5.6 Thiamine pyrophosphate (TPP) Thiamine pyrophosphate (also called thiamine diphosphate) is derived from vitamin B (thiamine) and has the structure: The thiazole ring can lose a proton to produce a negatively-charged carbon atom: (11.37) This is a potent nucleophile and can participate in covalent catalysis, particularly with u-keto (oxo) acid decarboxylase, u-keto acid oxidase, transketolase and phosphoketolase enzymes For example, pyruvate decarboxylase, found in yeast and some other microorganisms, utilizes TPP to catalyse the production of acetaldehyde from pyruvate Ronald Breslow (1957) proposed the following reaction mechanism: Sec 11.5] Coenzymes in enzyme-catalysed reactions 213 The actual decarboxylation step is facilitated by electrophilic catalysis as the thiazole ring withdraws electrons The reaction will proceed in the absence of enzyme, but the acetaldehyde formed tends to react with the TPP-C-(CH3)0H complex to produce acetoin as the final product It is likely that the enzyme stabilizes the TPP-acetaldehyde complex and prevents this condensation from occurring The pyruvate dehydrogenase multienzyme complex (see section 5.2.5) also catalyses the decarboxylation of pyruvate, but it utilizes a second coenzyme, lipoic acid, to introduce an oxidation step and a third coenzyme, coenzyme A (CoA.SH), to react with the acetyl-lipoamide complex, giving acetyl-CoA as the final product Initially, the TPP-C-(CH3)0H complex is formed as above, and then the reaction is thought to proceed as follows: CH3 CH3 R'-NAYR" CH,J:\ ~H S de:;:;:ase - CS V