121514382

Fundamentals of Enzyme Kinetics
Third edition
by Athel Cornish-Bowden
Universitats-und Landesbibliothek Bibliothek Biologie Schnittspahnstr. 10, 64287 Darmstadt
lnv,Nr.
PORTLAND PRESS
Contents
Preface to the Third Edition xv
Basic Principles of Chemical Kinetics

1.1 1.2 Abbreviations and symbols Order of a reaction 1.2.1 1.2.2 1.3 1.4 1.5 1.6 1.7 Order and molecularity Determination of the order of a reaction 1 2 2 6 8 9 10 12 13 13 15 16 20 21 .
Dimensions of rate constants Reversible reactions Determination of first-order rate constants Catalysis constants 1.7.1 1.7.2 1.7.3 1.7.4 The Arrhenius equation Elementary collision theory Transition-state theory Effects of hydrostatic pressure on rate constants The influence of temperature and pressure on rate
Problems
Introduction to Enzyme Kinetics

2.1 2.2 2.3 The idea of an enzyme-substrate complex The Michaelis-Menten equation The steady state of an enzyme-catalysed reaction 2.3.1 2.3.2 2.3.3 The Briggs-Haldane treatment The Michaelis-Menten equation Units of enzyme activity equation 2.3.5 2.3.6 2.4 Mutual depletion kinetics Ways of writing the Michaelis-Menten equation 23 25 29 29 30 31 32 34 35 36
2.3.4 The curve defined by the Michaelis-Menten
Specificity
Contents
2.5 2.6
Validity of the steady-state assumption Graphs of the Michaelis-Menten equation 2.6.1 2.6.2 2.6.3 2.6.4 2.6.5 Plotting v against a The double-reciprocal plot The plot of a/v against a The plot of v against via The direct linear plot The reversible rate equation The Haldane relationship "One-way enzymes"
39 41 41 42 44 45 46 48 48 52 53 55 57 57 58 60 63 64 64 65 66 67 68
2.7
The reversible Michaelis-Menten mechanism 2.7.1 2.7.2 2.7.3
2.8 2.9
Product inhibition Integration of enzyme rate equations 2.9.1 2.9.2 2.9.3 2.9.4 Michaelis-Menten equation without product inhibition Effect of product inhibition on the progress curve Accurate estimation of initial rates Time courses for other mechanisms
2.10
Artificial enzymes, RNA enzymes and catalytic antibodies 2.10.1 "Alternative enzymes" 2.10.2 Artificial enzymes 2.10.3 Catalytic RNA 2.10.4 Catalytic antibodies
Problems
Practical Aspects,of Kinetic Studies

3.1 Enzyme assays 3.1.1 3.1.2 3.1.3 Discontinuous and continuous assays Estimating the initial rate Increasing the straightness of the progress curve 71 71 72 73
3.1.4
3.2 3.3
Coupled assays
75
78 81 81 83 84 86 89
Detecting enzyme inactivation Experimental design 3.3.1 3.3.2 3.3.3 Choice of substrate concentrations Choice of pH, temperature and other conditions Use of replicate observations
3.4
Treatment of ionic equilibria ,
Problems
Contents
Deriving Steady-State Rate Equations

4.1 4.2 4.3 4.4 4.5 4.6 4.7 Introduction The principle of the King-Altman method The method of King and Altman The method of Wong and Hanes Modifications to the King-Altman method Reactions containing steps at equilibrium Analysing mechanisms by inspection 4.7.1 4.7.2 4.7.3 4.8 Problems Topological reasoning Mechanisms with alternative routes Dead-end steps
Derivation of rate equations by computer
91 91 95 99 100 103 106 106 107 107 108 111
Reversible Inhibition and Activation

5.1 5.2 Introduction Linear inhibition 5.2.1 5.2.2 5.2.3 ' 5.2.4 5.3 5.3.1 5.3.2 5.4 5.5 5.6 Competitive inhibition Mixed inhibition Uncompetitive inhibition Summary of linear inhibition types Simple plots Combination plots 113 114 114 116 119 120 121 121 124 125 126 127 127 128 130 130 131 133 135 137 ' 137 140 141
Plotting inhibition results
Multiple inhibitors Relationship between inhibition constants and the concentration for half-inhibition Inhibition by a competing substrate 5.6.1 5.6.2 Competition between substrates Testing if two reactions occur at the same site Miscellaneous uses of the term activation Essential activation Hyperbolic activation and inhibition
5.7
Enzyme activation 5.7.1 5.7.2 5.7.3
5.8 5.9
Design of inhibition experiments Inhibitory effects of substrates 5.9.1 5.9.2 Non-productive binding Substrate inhibition
Problems
Contents
Tight-binding and Irreversible Inhibitors

6.1 6.2 Tight-binding inhibitors Irreversible inhibitors 6.2.1 6.2.2 6.3 6.4 Non-specific irreversible inhibition Specific irreversible inhibition 145 148 148 148 150 151 151 153 155
Substrate protection experiments Chemical modification as a means of identifying essential groups 6.4.1 6.4.2 Kinetic analysis of chemical modification Remaining activity as a function of degree of modification
Problems
Reactions of More than One Substrate

7.1 7.2 Introduction Classification of mechanisms 7.2.1 7.2.2 7.2.3 7.2.4 7.3 7.3.1 7.3.2 7.3.3 7.3.4 7.4 Ternary-complex mechanisms Substituted-enzyme mechanisms Comparison between chemical and kinetic classifications Schematic representation of mechanisms Compulsory-order ternary-complex mechanism Random-order ternary-complex mechanism Substituted-enzyme mechanism Calculation of rate constants from kinetic parameters Initial-rate measurements in the absence of products 7.4.1 7.4.2 7.4.3 7.4.4 7.4.5 7.5 7.5.1 7.5.2 7.5.3 7.5.4 7.5.5 Meanings of the parameters Apparent Michaelis-Menten parameters Primary plots for ternary-complex mechanisms Secondary plots Plots for the substituted-enzyme mechanism Why substrate inhibition occurs Compulsory-order ternary-complex mechanism Random-order ternary-complex mechanism Substituted-enzyme mechanism Diagnostic value of substrate inhibition 170 171 171 173 174 175 177 178 178 179 179 180 180 Rate equations 163 165 166 166 169 169 157 158 158 161
Substrate inhibition
Contents
7.6 7.7 7.8
Product inhibition Design of experiments Reactions with three or more substrates
181 184 185 188
Problems
Use of Isotopes for Studying Enzyme Mechanisms

8.1 8.2 8.3 8.4 8.5 Isotope exchange and isotope effects Principles of isotope exchange Isotope exchange at equilibrium Isotope exchange in substituted-enzyme mechanisms Non-equilibrium isotope exchange 8.5.1 8.5.2 8.5.3 8.6 8.6.1 8.6.2 8.6.3 8.7 8.8 Chemiflux ratios Isomerase kinetics Tracer perturbation Primary isotope effects Secondary isotope effects Equilibrium isotope effects 191 192 194 196 197 197 200 203 204 204 206 207 207 209 211
Theory of kinetic isotope effects
Primary isotope effects in enzyme kinetics Solvent isotope effects
Problems
Effect of pH on Enzyme Activity

9.1 9.2 9.3 Enzymes and pH Acid-base properties of proteins lonization of a dibasic acid 9.3.1 9.3.2 9.3.3 9.4 9.4.1 9.4.2 9.4.3 9.4.4 9.4.5 9.5 Expression in terms of group dissociation constants Molecular dissociation constants Bell-shaped curves Underlying assumptions pH dependence of V and V/Km pH-independent parameters and their relationship to "apparent" parameters pH dependence of Km Experimental design 223 224 225 226 216 217 219 221 221 222 213 214 216
Effect of pH on enzyme kinetic constants
lonization of the substrate
Contents
9.6
More complicated pH effects
228 228
Problems
10 _
Temperature Effects on Enzyme Activity

10.1 10.2 10.3 10.4 Temperature denaturation Temperature optimum Application of the Arrhenius equation to enzymes Entropy-enthalpy compensation 231 233 234 235 237
Problems
11
Control of Enzyme Activity

11.1 Function of cooperative and allosteric interactions 11.1.1 Futile cycles '11.1.2 Inadequacy of Michaelis-Menten kinetics for regulation 11.1.3 Cooperativity 11.1.4 Allosteric interactions 11.2 The development of models to explain cooperativity 11.2.1 The Hill equation 11.2.2 An alternative index of cooperativity 11.2.3 Assumption of equilibrium binding in cooperative kinetics 11.2.4 The Adair equation 11.2.5 Mechanistic and operational definitions of cooperativity 11.3 Analysis of binding experiments 11.3.1 Equilibrium dialysis 11.3.2 The Scatchard plot 11.4 11.5 Induced fit The symmetry model of Monod, Wyman and Changeux 11.5.1 Basic postulates of the symmetry model 11.5.2 Algebraic analysis 11.5.3 Properties implied by the binding equation 11.5.4 Heterotropic effects 11.6 The sequential model of Koshland, Nemethy and Filmer 11.6.1 Postulates 11.6.2 Algebraic analysis 11.6.3 Properties implied by the binding equation 252 254 254 255 257 260 260 261 262 265 266 266 268 270 246 247 240 241 242 243 243 245 239 239
Contents
11.7 11.8
Association-dissociation models of cooperativity Kinetic cooperativity
272 273 275
Problems
12
Kinetics of Multi-Enzyme Systems

12.1 Enzymes in their physiological context 12.1.1 Enzymes as components of systems 12.1.2 Moiety conservation 12.1.3 Characterization of enzymes in permeabilized 277 277 278
cells
12.2 12.3 Metabolic control analysis Elasticities 12.3.1 Definition of elasticity 12.3.2 Common properties of elasticities 12.3.3 Enzyme kinetics viewed from control analysis 12.3.4 Rates and concentrations as effects, not causes 12.4 Control coefficients 12.4.1 Definitions 12.4.2 The perturbing parameter 12.5 12.6 Summation relationships Relationships between elasticities and control coefficients 12.6.1 Connectivity properties 12.6.2 Control coefficients in a three-step pathway 12.6.3 Expression of summation and connectivity relationships in matrix form 12.6.4 Connectivity relationship for a metabolite not involved in feedback 12.6.5 The flux control coefficient of an enzyme for the flux through its own reaction 12.7 12.8 12.9 Response coefficients: the partitioned response Control and regulation Mechanisms of regulation 12.9.1 Metabolite channelling 12.9.2 Interconvertible enzyme cascades 12.9.3 The metabolic role of adenylate kinase 12.10 Computer modelling of metabolic systems Problems
279
280 282 282 285 286 287 290 290 292 293 295 295 297 298 299 300 301 302 305 306 308 310 312 315
Contents
13
Fast Reactions
13.1 Limitations of steady-state measurements 13.1.1 The transient state 13.1.2 The relaxation time 13.1.3 "Slow" and "fast" steps in mechanisms 13.1.4 Ambiguities in the steady-state analysis of systems with intermediate isomerization 13.1.5 Ill-conditioning 13.2 Product release before completion of the catalytic cycle 13.2.1 "Burst" kinetics 13.2.2 Active site titration 13.3 Experimental techniques 13.3.1 Classes of method 13.3.2 Continuous flow 13.3.3 Stopped flow 13.3.4 Quenched flow 13.3.5 Flash photolysis 13.3.6 Magnetic resonance methods 13.3.7 Relaxation methods 13.4 Transient-state,kinetics 13.4.1 Systems far from equilibrium 13.4.2 Simplification of complicated mechanisms 13.4.3 Systems close to equilibrium Problems 320 321 323 323 325 326 326 328 329 330 332 334 334 336 336 340 343 345 317 317 318 318
14
Estimation of Kinetic Constants

14.1 14.2 The effect of experimental error on kinetic analysis Least-squares fit to the Michaelis-Menten equation 14.2.1 Introduction of error in the Michaelis-Menten equation 14.2.2 Estimation of the-Michaelis-Menten parameters 14.2.3 Corresponding results for a uniform standard deviation in the rates 14.3 Statistical aspects of the direct linear plot 14.3.1 Comparison between classical and distribution-free statistics 14.3.2 Application to the direct linear plot 14.3.3 Lack of need for weighting 355 357 358 354 355 350 352 347 350
Contents
14.3.4 Insensitivity to outliers 14.4 14.5
'
359 359 361 367 372
14.3.5 Handling of negative parameter estimates Precision of estimated kinetic parameters Residual plots and their uses
Problems References ,
375 399 407
Solutions and Notes to Problems Index

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Fundamentals of Enzyme Kinetics

Universitats-und Landesbibliothek Bibliothek Biologie Schnittspahnstr. 10, 64287 Darmstadt

Basic Principles of Chemical Kinetics

Introduction to Enzyme Kinetics

2.3.4 The curve defined by the Michaelis-Menten

The reversible Michaelis-Menten mechanism 2.7.1 2.7.2 2.7.3

Practical Aspects,of Kinetic Studies

Treatment of ionic equilibria ,

Deriving Steady-State Rate Equations

Derivation of rate equations by computer

91 91 95 99 100 103 106 106 107 107 108 111

Reversible Inhibition and Activation

Plotting inhibition results

Enzyme activation 5.7.1 5.7.2 5.7.3

Tight-binding and Irreversible Inhibitors

Reactions of More than One Substrate

7.6 7.7 7.8

Product inhibition Design of experiments Reactions with three or more substrates

181 184 185 188

Use of Isotopes for Studying Enzyme Mechanisms

Theory of kinetic isotope effects

Primary isotope effects in enzyme kinetics Solvent isotope effects

Effect of pH on Enzyme Activity

Effect of pH on enzyme kinetic constants

lonization of the substrate

More complicated pH effects

Temperature Effects on Enzyme Activity

Control of Enzyme Activity

Association-dissociation models of cooperativity Kinetic cooperativity

272 273 275

Kinetics of Multi-Enzyme Systems

Estimation of Kinetic Constants

14.3.4 Insensitivity to outliers 14.4 14.5

359 359 361 367 372

375 399 407

Solutions and Notes to Problems Index

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