Author's preface xvii Part 1: Structure and function of enzymes 1 An introduction to enzymes 1.1 What are enzymes 3 1.2 A brief history of enzymes 3 1.3 The naming and classification of enzymes 4 1.3.1 Why classify enzymes? 4 1.3.2 The Enzyme Commission's system of classification 5 1.3.3 The Enzyme Commission's recommendations on nomenclature 6 1.3.4 The six main classes of enzymes 6 Summary of Chapter 1 12 Further reading 12 Problems 12 2 The structure of proteins 2.1 Introduction 15 2.2 Amino acids, the building blocks of proteins 16 2.2.1 Structure and classification of amino acids 16 2.2.2 Stereochemistry of amino acids 18 2.3 The basis of protein structure 19 2.3.1 Levels of protein structure 19 2.3.2 Bonds involved in the maintenance of protein structure 21 2.4 The determination of primary structure 23 2.4.1 The isolation of each polypeptide chain 23 2.4.2 Determination of the amino acid composition of each polypeptide chain 26 2.4.3 Determination of the amino acid sequence of each polypeptide chain 27 vii
viii 2.4.4 Determination of the positions of disulphide bridges 30 2.4.5 Some results of experimental investigation of primary structure 30 2.4.6 Indirect determination of primary structure 31 2.5 The determination of protein structure by X-ray crystallography... 31 2.5.1 The principles of X-ray crystallography 31 2.5.2 Some results of X-ray crystallography 36 2.6 The investigation of protein structure in solution 40 Summary of Chapter 2 41 Further reading 42 Problems 42 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 56 3.2.1 Chemical properties of proteins 56 3.2.2 Acid-base properties of proteins 57 3.2.3 The solubility of globular proteins 61 Summary of Chapter 3 64 Further reading 65 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 74 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 77 5.2 Oligomeric enzymes 79 5.2.1 Introduction 79 5.2.2 Lactate dehydrogenase 79 5.2.3 Lactose synthase 81
ix 5.2.4 Tryptophan synthase 82 5.2.5 Pyruvate dehydrogenase 82 Summary of Chapter 5 84 Further reading 84 Part 2: Kinetic and chemical mechanisms of enzyme-catalysed reactions 6 An introduction to bioenergetics, catalysis and kinetics 6.1 Some concepts of bioenergetics 87 6.1.1 The first and second laws of thermodynamics 87 6.1.2 Enthalpy, entropy and free energy 87 6.1.3 Free energy and chemical reactions 88 6.1.4 Standard free energy 89 6.1.5 Bioenergetics and the living cell 90 6.2 Factors affecting the rates of chemical reactions 91 6.2.1 The collision theory 91 6.2.2 Activation energy and the transition-state theory 91 6.2.3 Catalysis 94 6.3 Kinetics of uncatalysed chemical reactions 95 6.3.1 The Law of Mass Action and the order of reaction 95 6.3.2 The use of initial velocity 97 6.4 Kinetics of enzyme-catalysed reactions: an historical introduction... 98 6.5 Methods used for investigating the kinetics of enzyme-catalysed reactions 100 6.5.1 Initial velocity studies 100 6.5.2 Rapid-reaction techniques 102 6.6 The nature of enzyme catalysis 103 Summary of Chapter 6 104 Further reading 105 Problems 105 7 Kinetics of single-substrate enzyme-catalysed reactions 7.1 The relationship between initial velocity and substrate concentration 107 7.1.1 The Henri and Michaelis-Menten equations 107 7.1.2 The Briggs-Haldane modification of the Michaelis-Menten equation 109 7.1.3 The significance of the Michaelis-Menten equation Ill 7.1.4 The Lineweaver-Burk plot 113 7.1.5 The Eadie-Hofstee and Hanes plots 114 7.1.6 The Eisenthal and Cornish-Bowden plot 116 7.1.7 The Haldane relationship for reversible reactions 117 7.2 Rapid-reaction kinetics 118 7.2.1 Pre-steady-state kinetics 118 7.2.2 Relaxation kinetics 122 7.3 The King and Altman procedure 124 Summary of Chapter 7 126
x Further reading 126 Problems 127 8 Enzyme inhibition 8.1 Introduction 128 8.2 Reversible inhibition 128 8.2.1 Competitive inhibition 128 8.2.2 Uncompetitive inhibition 135 8.2.3 Non-competitive inhibition 138 8.2.4 Mixed inhibition 142 8.2.5 Partial inhibition 146 8.2.6 Substrate inhibition 146 8.2.7 Allosteric inhibition 148 8.3 Irreversible inhibition 149 Summary of Chapter 8 151 Further reading 152 Problems 152 9 Kinetics of multi-substrate enzyme-catalysed reactions 9.1 Examples of possible mechanisms 155 9.1.1 Introduction 155 9.1.2 Ping-pong bi-bi mechanism 155 9.1.3 Random-order mechanism 156 9.1.4 Compulsory-order mechanism 156 9.2 Steady-state kinetics 157 9.2.1 The general rate equation of Alberty 157 9.2.2 Plots for mechanisms which follow the general rate equation 158 9.2.3 The general rate equation of Dalziel 159 9.2.4 Rate constants and the constants of Alberty and Dalziel... 160 9.3 Investigation of reaction mechanisms using steady-state methods... 162 9.3.1 The use of primary plots 162 9.3.2 The use of inhibitors which compete with substrates for binding sites 163 9.4 Investigation of reaction mechanisms using non-steady-state methods 167 9.4.1 Isotope exchange at equilibrium 167 9.4.2 Rapid-reaction studies 169 Summary of Chapter 9 169 Further reading 170 Problems 170 10 The investigation of active site structure 10.1 The identification of binding sites and catalytic sites 175 10.1.1 Trapping the enzyme-substrate complex 175 10.1.2 The use of substrate analogues 176 10.1.3 Enzyme modification by chemical procedures affecting
xi amino acid side chains 177 10.1.4 Enzyme modification by treatment with proteases 181 10.1.5 Enzyme modification by site-directed mutagenesis 181 10.1.6 The effect of changing ph 182 10.2 The investigation of the three-dimensional structures of active sites 187 Summary of Chapter 10 189 Further reading 189 Problems 190 11 The chemical nature of enzyme catalysis 11.1 An introduction to reaction mechanisms in organic chemistry 191 11.2 Mechanisms of catalysis... 193 11.2.1 Acid-base catalysis 193 11.2.2 Electrostatic catalysis 194 11.2.3 Covalent catalysis 194 11.2.4 Enzyme catalysis! 195 11.3 Mechanisms of reactions catalysed by enzymes without cofactors.. 196 11.3.1 Introduction 196 11.3.2 Chymotrypsin 196 11.3.3 Ribonuclease 198 11.3.4 Lysozyme 199 11.3.5 Triose phosphate isomerase 201 11.4 Metal-activated enzymes and metalloenzymes 202 11.4.1 Introduction 202 11.4.2 Activation by alkali metal cations (Na + and K + ) 202 11.4.3 Activation by alkaline earth metal cations (Ca 2+ and Mg 2+ ) 203 11.4.4 Activation by transition metal cations (Cu, Zn, Mo, Fe and Co cations) 204 11.5 The involvement of coenzymes in enzyme-catalysed reactions 206 11.5.1 Introduction 206 11.5.2 Nicotinamide nucleotides (NAD + and NADP + ) 207 11.5.3 Flavin nucleotides (FMN and FAD) 209 11.5.4 Adenosine phosphates (ATP, ADP and AMP) 212 11.5.5 Coenzyme A (CoA.SH) 213 11.5.6 Thiamine pyrophosphate (TPP) 214 11.5.7 Pyridoxal phosphate 215 11.5.8 Biotin 217 11.5.9 Tetrahydrofolate 218 11.5.10 Coenzyme B 12 220 Summary of Chapter 11 222 Further reading 222 12 The binding of ligands to proteins 12.1 Introduction 223 12.2 The binding of a ligand to a protein having a single ligand-binding site 223
xii 12.3 Cooperativity 224 12.4 Positive homotropic cooperativity and the Hill equation 225 12.5 The Adair equation for the binding of a ligand to a protein having two binding sites for that ligand 228 12.5.1 General considerations 228 12.5.2 Where there is no interaction between the binding sites... 229 12.5.3 Where there is positive homotropic cooperativity 231 12.5.4 Where there is negative homotropic cooperativity 231 12.6 The Adair equation for the binding of a ligand to a protein having three binding sites for that ligand 232 12.7 The Adair equation for the binding of a ligand to a protein having four binding sites for that ligand 233 12.8 Investigation of cooperative effects 233 12.8.1 Measurement of the relationship between Y and [S] 233 12.8.2 Measurement of the relationship between v 0 and [S o ] 234 12.8.3 The Scatchard plot and equilibrium dialysis techniques... 234 12.9 The binding of oxygen to haemoglobin 237 Summary of Chapter 12 238 Further reading 238 Problems 239 13 Sigmoidal kinetics and allosteric enzymes 13.1 Introduction 240 13.2 The Monod-Wyman-Changeux (MWC) model 240 13.2.1 The MWC equation 240 13.2.2 How the MWC model accounts for cooperative effects... 243 13.2.3 The MWC model and allosteric regulation 243 13.2.4 The MWC model and the Hill equation 245 13.3 The Koshland-Némethy-Filmer (KNF) model 246 13.3.1 The KNF model for a dimeric protein 246 13.3.2 The KNF model for any oligomeric enzyme 248 13.3.3 The KNF model and allosteric regulation 249 13.4 Differentiation between models for cooperative binding in proteins 249 13.5 Sigmoidal kinetics in the absence of cooperative binding 250 13.5.1 Ligand-binding evidence versus kinetic evidence 250 13.5.2 The Ferdinand mechanism 250 13.5.3 The Rabin and mnemonical mechanisms 251 Summary of Chapter 13 252 Further reading 252 Problems 253 14 The significance of sigmoidal behaviour 14.1 The physiological importance of cooperative oxygen-binding by haemoglobin 257 14.2 Allosteric enzymes and metabolic regulation 258 14.2.1 Introduction 258
xiii 14.2.2 Characteristics of steady-state metabolic pathways 259 14.2.3 Regulation of steady-state metabolic pathways by control of enzyme activity 261 14.2.4 Allosteric enzymes and the amplification of metabolic regulation 263 14.2.5 Other mechanisms of metabolic regulation 263 14.2.6 Some examples of allosteric enzymes involved in metabolic regulation 269 Summary of Chapter 14 272 Further reading 273 Part 3: Application of enzymology 15 Investigation of enzymes in biological preparations 15.1 Choice of preparation for the investigation of enzyme characteristics 277 15.2 Enzyme assay 279 15.2.1 Introduction 279 15.2.2 Enzyme assay by kinetic determination of catalytic activity 279 15.2.3 Coupled kinetic assays 284 15.2.4 Radioimmunoassay (RIA) of enzymes 286 15.3 Investigation of sub-cellular compartmentation of enzymes 287 15.3.1 Enzyme histochemistry 287 15.3.2 The use of centrifugation 288 15.3.3 Some results of the investigation of enzyme compartmentation 291 Summary of Chapter 15 292 Further reading 293 Problem 293 16 Extraction and purification of enzymes 16.1 Extraction of enzymes 295 16.1.1 Introduction 295 16.1.2 The extraction of soluble enzymes 295 16.1.3 The extraction of membrane-bound enzymes 296 16.1.4 The nature of the extraction medium 299 16.2 Purification of enzymes 300 16.2.1 Preliminary purification procedures 300 16.2.2 Further purification procedures 301 16.2.3 Criteria of purity 305 16.3 Determination of molecular weights of enzymes 308 Summary of Chapter 16 309 Further reading : 310 Problem 310 17 Enzymes as analytical reagents 17.1 The value of enzymes as analytical reagents 312
xiv 17.2 Principles of enzymatic analysis 313 17.2.1 End-point methods 313 17.2.2 Kinetic methods 316 17.2.3 Immunoassay methods 320 17.3 Handling enzymes and coenzymes 321 Summary of Chapter 17 322 Further reading 323 Problems 323 18 Instrumental techniques available for use in enzymatic analysis 18.1 Principles of the available detection techniques 324 18. 1.1 18. 1.2 18. 1.3 18. 1.4 18. 1.5 18. L.6 18. L.7 18. 1.8 Introduction Manometry Spectrophotometry Spectrofluorimetry Electrochemical methods Enthalpimetry Radiochemical methods Dry-reagent techniques 324 324 325 326 327 330 331 331 18.2 Automation in enzymatic analysis 332 18.2.1 Introduction 332 18.2.2 Fixed-time methods 333 18.2.3 Fixed-concentration methods 337 18.2.4 Methods involving continuous monitoring 337 Summary of Chapter 18 339 Further reading 339 19 Some applications of enzymatic analysis in medicine and industry 19.1 Applications in medicine 340 Q9.1.J! Assay of plasma enzymes 340 19.1.2 Enzymes and inborn errors of metabolism 345 19.1.3 Enzymes as reagents in clinical chemistry 349 19.2 Applications in industry 349 Summary of Chapter 19 351 Further reading 351 20 Biotechnological applications of enzymes 20.1 Large-scale production and purification of enzymes 352 20.1.1 Production of enzymes on an industrial scale 352 20.1.2 Large-scale purification of enzymes 354 20.1.3 Synthesis of artificial enzymes 355 20.2 Immobilized enzymes 356 20.2.1 Preparation of immobilized enzymes 356 20.2.2 Properties of immobilized enzymes 362 20.2.3 Applications of immobilized enzymes: general principles 20.3 Enzyme utilization in industry 363 20.3.1 Introduction 365
Table of Contents xv 20.3.2 Applications in food and drink industries 366 20.3.3 Applications in other industries 368 20.4 Enzymes and recombinant DNA technology 369 20.4.1 Introduction 369 20.4.2 Applications 371 20.5 Enzymes and bioinformatics 379 Summary of Chapter 20 380 Further reading 381 Problems 384 Answers to problems 386 Abbreviations 391 Index 393