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 do 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 of Samuel 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 single- author 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. 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British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library ISBN 978-1-904275-27-5 Printed by Lightning Source Table of Contents Authors' preface ............................................................................. xiv Part 1 : Structure and function of enzymes 1 An introduction to enzymes 1.1 What are enzymes? ...................................................................................... 2 1.2 A brief history of enzymes .......................................................................... 2 1.3 The naming and classification of enzymes ................................................. 3 1.3 .1 Why classify enzymes? ................................................................... 3 1.3 .2 The Enzyme Commission's system of classification ....................... 4 1.3.3 The Enzyme Commission's recommendations on nomenclature .................................................................................. 5 1.3.4 The six main classes of enzymes ..................................................... 6 Summary of Chapter 1 .................................................................................... 11 Further reading ................................................................................................ 11 Problems .......................................................................................................... 11 2 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 2 .................................................................. 42 Further reading ........................................................................... 42 Problems ................................................................................. 43 3 The biosynthesis and properties of proteins 3 .1 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 3 .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 3 .................................................................. 64 Further reading .......................................................................... 64 Problems .................................................................................. 65 4 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 4 ................................................................... 74 Further reading ............................................................................. 75 5 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 5 .................................................................. 83 Further reading ........................................................................... 83 Table of Contents vii Part 2 : Kinetic and chemical mechanisms of enzyme-catalysed reactions 6 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 6 .................................................................. 102 Further reading ......................................................................... 102 Problems ................................................................................. 102 7 Kinetics of single-substrate enzyme-catalysed reactions 7 .1 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 7 .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 7 .2 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 7 ................................................................. 124 Further reading ......................................................................... 124 Problems ................................................................................ 125 viii Table of Contents 8 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 8 ................................................................ 149 Further reading ........................................................................ 150 Problems ................................................................................ 150 9 Kinetics of multi-substrate enzyme-catalysed reactions 9.1 Examples of possible mechanisms ............................................. 153 9.1.1 Introduction ............................................................... 153 9 .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 9 .3 Investigation of reaction mechanisms using steady-state methods ........ 160 9.3.1 The use of primary plots ................................................ 160 9 .3 .2 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 9 ......................................................... 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 ....................................... 17 4 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 B12 ......................................................... 218 Summary of Chapter 11 .............................................................. 220 Further reading ......................................................................... 220 12 The binding of ligands to proteins 12.1 Introduction ...................................................................... 222 x Table of Contents 12.2 The binding of a ligand to a protein having a single ligand-binding site .................................................................................. 222 12.3 Cooperativity .................................................................... 223 12.4 Positive homotropic cooperativity and the Hill equation .................. 224 12.5 The Adair equation for the binding of a ligand to a protein having two binding sites for that ligand ................................... 227 12.5.1 General considerations ............................................... 227 12.5.2 Where there is no interaction between the binding sites ........ 228 12.5.3 Where there is positive homotropic cooperativity .............. 230 12.5.4 Where there is negative homotropic cooperativity .............. 230 12.6 The Adair equation for the binding of a ligand to a protein having three binding sites for that ligand .................................. 231 12. 7 The Adair equation for the binding of a ligand to a protein having four binding sites for that ligand .................................... 232 12.8 Investigation of cooperative effects .......................................... 232 12.8.1 Measurement of the relationship between Y and (S] ............ 232 12.8.2 Measurement of the relationship between v0 and [So] ............ 233 12.8.3 The Scatchard plot and equilibrium dialysis techniques ........ 233 12.9 The binding ofoxygen to haemoglobin ..................................... 236 SummaryofChapter 12 ............................................................. 237 Further reading ......................................................................... 237 Problems ................................................................................ 238 13 Sigmoidal kinetics and allosteric enzymes 13 .1 Introduction ....................................................................... 23 9 13 .2 The Monod-Wyman-Changeux (MWC) model ............................ 239 13.2.1 The MWC equation .................................................. 239 13.2.2 How the MWC model accounts for cooperative effects ......... 242 13.2.3 The MWC model and allosteric regulation ....................... 242 13.2.4 The MWC model and the Hill equation ........................... 244 13.3 The Koshland-Nemethy-Filmer (KNF) model .............................. 245 13.3.1 The KNF model for a dimeric protein ............................. 245 13.3.2 The KNF model for any oligomeric enzyme ..................... 247 13 .3 .3 The KNF model and allosteric regulation ......................... 248 13 .4 Differentiation between models for cooperative binding in proteins ...................................................................... 248 13.5 Sigmoidal kinetics in the absence of cooperative binding ................ 249 13.5.1 Ligand-binding evidence versus kinetic evidence ................ 249 13.5.2 The Ferdinand mechanism ........................................... 250 13.5.3 The Rabin and mnemonical mechanisms .......................... 250 Summary of Chapter 13 .............................................................. 251 Further reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... 251 Problems ............................................................................... 252 Table of Contents xi 14 The significance of sigmoidal behaviour 14.1 The physiological importance of cooperative oxygen-binding by haemoglobin ............................................................... 255 14.2 Allosteric enzymes and metabolic regulation ................................ 257 14.2.1 Introduction ............................................................ 257 14.2.2 Characteristics of steady-state metabolic pathways ............... 258 14.2.3 Regulation of steady-state metabolic pathways by control of enzyme activity ........................................ 260 14.2.4 Allosteric enzymes and the amplification of metabolic regulation ............................................................. 262 14.2.5 Other mechanisms of metabolic regulation ........................ 263 14.2.6 Some examples of allosteric enzymes involved in metabolic regulation ................................................ 268 Summary of Chapter 14 ............................................................ 271 Further reading ....................................................................... 272 Part 3: Application of enzymology 15 Investigation of enzymes in biological preparations 15 .1 Choice of preparation for the investigation of enzyme characteristics ................................................................. 274 15.2 Enzyme assay ................................................................... 276 15.2.l Introduction ............................................................ 276 15 .2.2 Enzyme assay by kinetic determination of catalytic activity ............................................................... 277 15.2.3 Coupled kinetic assays ............................................... 280 15.2.4 Radioimmunoassay (RIA) of enzymes ............................. 282 15 .3 Investigation of sub-cellular compartmentation of enzymes .............. 284 15. 3 .1 Enzyme histochemistry ............................................... 2 84 15 .3 .2 The use of centrifugation ............................................. 286 15.3.3 Some results of the investigation of enzyme compartmentation .................................................. 289 Summary of Chapter 15 .................................................................. 291 Further reading ...................................................................... 291 Problem ................................................................................ 292 16 Extraction and purification of enzymes 16.1 Extraction of enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 293 16.1.1 Introduction ................................................... ...293 16.1.2 The extraction of soluble enzymes . . .. . . . . . . . . . .. . . . . . . . . . . .. 293 16.1.3 The extraction of membrane-bound enzymes ............ . .. 294 16.1.4 The nature of the extraction medium ........................ . .. 297 16.2 Purification of enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. 298 16.2.1 Preliminary purification procedures .............................. 298 16.2.2 Further purification procedures .................................... 299 16.2.3 Criteria of purity ..................................................... 307 xii Table of Contents 16.3 Determination of molecular weights of enzymes ................................ .311 Summary of Chapter 16 .............................................................. 312 Furtherreading ........................................................................ 313 Problem ............................................................................... .314 17 Enzymes as analytical reagents 17 .1 The value of enzymes as analytical reagents ................................. 315 17 .2 Principles of enzymatic analysis .............................................. 316 17.2.l End-point methods ................................................... 316 17.2.2 Kinetic methods ...................................................... 319 17.2.3 Immunoassay methods ............................................. 323 17.3 Handling enzymes and coenzymes ......................................... 324 Summary of Chapter 17 ............................................................ .326 Further reading ...................................................................... .326 Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .327 18 Instrumental techniques available for use in enzymatic analysis 18.1 Principles of the available detection techniques .......................... .328 18.1.1 Introduction ........................................................... 328 18.1.2 Manometry ............................................................ 328 18.1.3 Spectrophotometry ................................................... 329 18.1.4 Spectrofluorimetry ................................................... 330 18.1.5 Electrochemical methods ........................................... 331 18.1.6 Enthalpimetry ......................................................... 334 18.1. 7 Radiochemical methods ............................................. 334 18.1.8 Dry-reagent techniques .............................................. 335 18.2 Automation in enzymatic analysis ............................................ 336 18.2.1 Introduction .......................................................... 336 18.2.2 Fixed-time methods ................................................... 33 7 18.2.3 Fixed-concentration methods ....................................... 339 18.2.4 Methods involving continuous monitoring ........................ 340 18.3 High-throughput assays (HTA) ............................................... 341 Summary of Chapter 18 ............................................................. 342 Further reading ........................................................................ 342 19 Applications of enzymatic analysis in medicine, forensic science and industry 19.1 Applications in medicine ....................................................... 343 19 .1.1 Assay of plasma enzymes ............................................ 343 19 .1.2 Enzymes and inborn errors of metabolism ....................... 348 19.1.3 Enzymes as reagents in clinical chemistry ........................ 352 19.2 Applications in forensic science .............................................. 352 19.3 Applications in industry ....................................................... 353 Summary of Chapter 19 .............................................................. 355 Further reading ....................................................................... 355 Table of Contents Xlll 20 Biotechnological applications of enzymes 20.1 Large-scale production and purification of enzymes ....................... 356 20.1.1 Production of enzymes on an industrial scale .................... 356 20.1.2 Large-scale purification of enzymes ............................... 359 20.1.3 Synthesis of artificial enzymes ..................................... 360 20.2 Immobilized enzymes .......................................................... 361 20.2.1 Preparation of immobilized enzymes .............................. 361 20.2.2 Properties of immobilized enzymes ................................ 366 20.2.3 Applications of immobilized enzymes: general principles ...................................................... .368 20.3 Enzyme utilization in industry ................................................ 369 20.3.1 Introduction ........................................................... 369 20.3.2 Applications in food and drink industries ......................... 370 20.3.3 Applications in other industries ..................................... 373 Summary of chapter 20 ............................................................. 374 Further reading ....................................................................... 374 Problems ............................................................................... 376 21 Genomics, proteomics and bioinformatics 21.1 Enzymes and recombinant DNA technology ................................ 377 21.1.1 Introduction ........................................................... 3 77 21.1.2 Applications .......................................................... 378 21.2 Proteomics ....................................................................... 387 21.2.1 The application of mass spectrometry to the investigation of the proteome ........................................................ 387 21.2.2 Proteomics research .................................................. 389 21.3 Enzymes and bioinformatics ................................................. 390 21.3.l Introduction .......................................................... 390 21.3.2 Systems biology and microarrays .................................. 392 Summary of Chapter 21 ............................................................ 393 Further reading ....................................................................... 393 Problems ............................................................................... 396 Answers to problems ........................................................................ 397 Abbreviations .............................................................................. 403 Index .......................................................................................... 405 Authors' Preface This book was written, as all textbooks should be, with the requirements of the student firmly in mind. It is intended to provide an informative introduction to enzymology, and to give a balanced, reasonably-detailed, account of all the various theoretical and applied aspects of the subject which are likely to be included in an honours degree course. Furthermore, some of the later chapters may serve as a bridge to more advanced texts for students wishing to proceed further in this area of biochemistry. Although the book is intended mainly for students taking first degree courses which have a substantial biochemistry component, large portions may be of value to students on comparable courses in biological sciences, biomedical sciences or forensic sciences, and even to ones emolled on, in one direction, foundation programmes, or, in the other, MSc or other advanced courses who are approaching the subject of enzymology for the first time (or the first time in many years). No previous knowledge of biochemistry, and little of chemistry, is assumed. Most scientific terms are defined and placed in context when they are first introduced. Enzymology inevitably involves a certain amount of elementary mathematics, and some of the equations which are derived may appear somewhat complicated at first sight; however, once the initial biochemical assumptions have been understood, the derivations usually follow on the basis of simple logic, without involving any difficult mathematical manipulations. Numerical and other problems (with answers) are included, to test and reinforce the student's grasp of certain points. These problems generally use hypothetical data, although the results are often based on findings reported in the biochemical literature. If the size of the book is to be kept reasonable, some things of value have to be left out. The chief aim of this particular book is to help the student understand the concepts involved in enzymology, and the historical context in which they were worked out. It is not a reference book for practising enzymologists, so no comprehensive tables of data or long, finely-detailed accounts are included. Instead, an attempt has been made to give a perspective of each topic, and examples are quoted where appropriate. Credit has been given wherever possible to those responsible for the development of the subject, but many names deserving of mention have been excluded for reasons of space.