Fundamentals of Enzyme Kinetics
Bearbeitet von
Athel Cornish-Bowden
4., vollst. überarb. u. stark erw. Aufl. 2012. Taschenbuch. XVIII, 498 S. Paperback
ISBN 978 3 527 33074 4
Format (B x L): 17 x 24 cm
Gewicht: 985 g
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Contents
1
Basic Principles of Chemical Kinetics
1.1
Symbols, terminology and abbreviations
1.2
Order of a reaction
1.2.1
Order and molecularity
1.2.2
First-order kinetics
1.2.3
Second-order kinetics
1.2.4
Third-order kinetics
1.2.5
Zero-order kinetics
1.2.6
Determination of the order of a reaction
1.3
Dimensions of rate constants
1.4
Reversible reactions
1.5
Determination of first-order rate constants
1.6
The steady state
1.7
Catalysis
1.8
The influence of temperature and pressure on rate constants
1.8.1
The Arrhenius equation
1.8.2
Elementary collision theory
1.8.3
Transition-state theory
1.8.4
Effects of hydrostatic pressure
Chapter summary
Problems
1
1
3
3
4
5
7
8
8
9
10
11
13
14
15
15
17
18
21
22
23
2
Introduction to Enzyme Kinetics
2.1
The idea of an enzyme–substrate complex
2.2
The Michaelis–Menten equation
2.3
The steady state of an enzyme-catalyzed reaction
2.3.1
The Briggs–Haldane treatment
2.3.2
Parameters of the Michaelis–Menten equation
2.3.3
Units of enzyme activity
2.3.4
The curve defined by the Michaelis–Menten equation
25
25
28
32
32
33
34
35
vi
Contents
2.3.5
Mutual depletion kinetics
2.3.6
Ways of writing the Michaelis–Menten equation
2.4
Specificity
2.4.1
The fundamental property of enzymes
2.4.2
Discrimination between mixed substrates
2.4.3
Comparing different catalysts
2.5
Validity of the steady-state assumption
2.6
Graphs of the Michaelis–Menten equation
2.6.1
Plotting v against a
2.6.2
The double-reciprocal plot
2.6.3
The plot of a/v against a
2.6.4
The plot of v against v/a
2.6.5
Origins of the plots
2.6.6
The direct linear plot
2.7
The reversible Michaelis–Menten mechanism
2.7.1
The reversible rate equation
2.7.2
The Haldane relationship
2.7.3
“One-way enzymes”
2.8
Product inhibition
2.9
Integration of enzyme rate equations
2.9.1
Michaelis–Menten equation without product inhibition
2.9.2
Effect of product inhibition on progress curves
2.9.3
Other problems with time courses
2.9.4
Characterizing mutant enzymes
2.9.5
Accurate estimation of initial rates
2.9.6
Time courses for other mechanisms
Chapter summary
Problems
37
37
38
38
39
42
43
45
45
47
48
49
51
51
54
54
58
59
61
63
63
65
66
67
67
71
72
73
3
“Alternative” enzymes
3.1
Introduction
3.2
Artificial enzymes
3.3
Site-directed mutagenesis
3.4
Chemical mimics of enzyme catalysis
3.5
Catalytic RNA
3.6
Catalytic antibodies
Chapter summary
Problems
77
77
78
80
81
81
83
84
84
4
Practical Aspects of Kinetics
4.1
Enzyme assays
4.1.1
Discontinuous and continuous assays
4.1.2
Estimating the initial rate
4.1.3
Increasing the straightness of the progress curve
4.1.4
Coupled assays
85
85
85
86
88
89
Contents
vii
4.2
4.3
Detecting enzyme inactivation
Experimental design
4.3.1
Choice of substrate concentrations
4.3.2
Choice of pH, temperature and other conditions
4.3.3
Use of replicate observations
4.4
Treatment of ionic equilibria
Chapter summary
Problems
93
95
95
98
99
102
105
106
5
Deriving Steady-state Rate Equations
5.1
Introduction
5.2
The principle of the King–Altman method
5.3
The method of King and Altman
5.4
The method of Wong and Hanes
5.5
Modifications to the King–Altman method
5.6
Reactions containing steps at equilibrium
5.7
Analyzing mechanisms by inspection
5.7.1
Topological reasoning
5.7.2
Mechanisms with alternative routes
5.7.3
Dead-end steps
5.7.4
Undetectability of some isomerization steps
5.8
A simpler method for irreversible reactions
5.9
Derivation of rate equations by computer
Chapter summary
Problems
107
107
108
113
116
117
119
122
122
123
124
125
126
128
131
132
6
Reversible Inhibition and Activation
6.1
Introduction
6.2
Linear inhibition
6.2.1
Competitive inhibition
6.2.2
Mixed inhibition
6.2.3
Uncompetitive inhibition
6.2.4
Summary of linear inhibition types
6.3
Plotting inhibition results
6.3.1
Simple plots
6.3.2
Combination plots
6.4
Multiple inhibitors
6.5
Relationship between inhibition constants and the concentration for 50%
inhibition
6.6
Inhibition by a competing substrate
6.6.1
Competition between substrates
6.6.2
Testing if two reactions occur at the same site
6.7
Enzyme activation
6.7.1
Miscellaneous uses of the term activation
6.7.2
Essential activation
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136
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140
140
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149
149
150
152
152
153
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Contents
6.7.3
Mixed activation
6.7.4
Hyperbolic activation and inhibition
6.8
Design of inhibition experiments
6.9
Inhibitory effects of substrates
6.9.1
Nonproductive binding
6.9.2
Substrate inhibition
Chapter summary
Problems
155
155
157
159
159
162
164
165
7
Tight-binding and Irreversible Inhibitors
7.1
Tight-binding inhibitors
7.2
Irreversible inhibitors
7.2.1
Nonspecific irreversible inhibition
7.2.2
Specific irreversible inhibition
7.3
Substrate protection experiments
7.4
Mechanism-based inactivation
7.5
Chemical modification as a means of identifying essential groups
7.5.1
Kinetic analysis of chemical modification
7.5.2
Remaining activity as a function of degree of modification
7.6
Inhibition as the basis of drug design
7.7
Delivering a drug to its target
Chapter summary
Problems
169
169
172
172
173
174
175
179
180
181
183
186
187
188
8
Reactions of More than One Substrate
8.1
Introduction
8.2
Classification of mechanisms
8.2.1
Ternary-complex mechanisms
8.2.2
Substituted-enzyme mechanisms
8.2.3
Comparison between chemical and kinetic classifications
8.2.4
Schematic representation of mechanisms
8.3
Rate equations
8.3.1
Compulsory-order ternary-complex mechanism
8.3.2
Random-order ternary-complex mechanism
8.3.3
Substituted-enzyme mechanism
8.3.4
Haldane relationships
8.3.5
Calculation of rate constants from kinetic parameters
8.4
Initial-rate measurements in the absence of products
8.4.1
Meanings of the parameters
8.4.2
Apparent Michaelis–Menten parameters
8.4.3
Primary plots for ternary-complex mechanisms
8.4.4
Secondary plots
8.4.5
Plots for the substituted-enzyme mechanism
8.5
Substrate inhibition
8.5.1
Why substrate inhibition occurs
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189
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190
193
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197
198
198
200
202
202
203
204
204
207
208
209
210
211
211
Contents
9
ix
8.5.2
Compulsory-order ternary-complex mechanism
8.5.3
Random-order ternary-complex mechanism
8.5.4
Substituted-enzyme mechanism
8.5.5
Diagnostic value of substrate inhibition
8.6
Product inhibition
8.7
Design of experiments
8.8
Reactions with three or more substrates
8.8.1
Quaternary-complex mechanisms
8.8.2
Substituted-enzyme mechanisms
8.8.3
Hybrid mechanisms
8.8.4
Classification of three-substrate mechanisms
Chapter summary
Problems
211
212
212
213
213
217
218
219
219
220
221
223
224
Use of Isotopes for Studying Enzyme Mechanisms
9.1
Isotope exchange and isotope effects
9.2
Principles of isotope exchange
9.3
Isotope exchange at equilibrium
9.4
Isotope exchange in substituted-enzyme mechanisms
9.5
Nonequilibrium isotope exchange
9.5.1
Chemiflux ratios
9.5.2
Isomerase kinetics
9.5.3
Tracer perturbation
9.6
Theory of kinetic isotope effects
9.6.1
Primary isotope effects
9.6.2
Secondary isotope effects
9.6.3
Equilibrium isotope effects
9.7
Primary isotope effects in enzyme kinetics
9.8
Solvent isotope effects
Chapter summary
Problems
227
227
228
231
233
234
234
238
240
242
242
245
246
246
248
251
252
10 Effect of pH on Enzyme Activity
10.1 Enzymes and pH
10.2 Acid–base properties of proteins
10.3 Ionization of a dibasic acid
10.3.1 Expression in terms of group dissociation constants
10.3.2 Molecular dissociation constants
10.3.3 Bell-shaped curves
10.4 Effect of pH on enzyme kinetic constants
10.4.1 Underlying assumptions
10.4.2 pH dependence of V and V/Km
10.4.3 pH-independent parameters and their relationship to “apparent”
parameters
10.4.4 pH dependence of Km
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253
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257
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261
262
264
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Contents
10.4.5 Experimental design
10.5 Ionization of the substrate
10.6 “Crossed-over” ionization
10.7 More complicated pH effects
Chapter summary
Problems
266
268
268
269
269
270
11 Temperature Effects on Enzyme Activity
11.1 Temperature denaturation
11.2 Irreversible denaturation
11.3 Temperature optimum
11.4 Application of the Arrhenius equation to enzymes
11.5 Entropy–enthalpy compensation
Chapter summary
Problems
273
273
275
275
276
278
279
280
12 Regulation of Enzyme Activity
12.1 Function of cooperative and allosteric interactions
12.1.1 Futile cycles
12.1.2 Inadequacy of Michaelis–Menten kinetics for regulation
12.1.3 Cooperativity
12.1.4 Allosteric interactions
12.2 The development of models for cooperativity
12.2.1 The Hill equation
12.2.2 Specificity of non-Michaelis–Menten enzymes
12.2.3 An alternative index of cooperativity
12.2.4 Assumption of equilibrium binding in cooperative kinetics
12.2.5 The Adair equation
12.2.6 Mechanistic and operational definitions of cooperativity
12.3 Analysis of binding experiments
12.3.1 Equilibrium dialysis
12.3.2 The Scatchard plot
12.4 Induced fit
12.4.1 Enzyme specificity
12.4.2 Induced fit today
12.5 The symmetry model of Monod, Wyman and Changeux
12.5.1 Basic postulates of the symmetry model
12.5.2 Algebraic analysis
12.5.3 Properties implied by the binding equation
12.5.4 Heterotropic effects
12.6 Comparison between the principal models of cooperativity
12.7 The sequential model of Koshland, Némethy and Filmer
12.7.1 Postulates
12.7.2 Algebraic analysis
12.7.3 Properties implied by the binding equation
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288
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291
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297
297
298
302
302
304
304
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312
313
313
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318
Contents
12.8 Association-dissociation models of cooperativity
12.9 Kinetic cooperativity
Chapter summary
Problems
13 Multienzyme Systems
13.1 Enzymes in their physiological context
13.1.1 Enzymes as components of systems
13.1.2 Moiety conservation
13.1.3 Enzymes in permeabilized cells
13.2 Metabolic control analysis
13.3 Elasticities
13.3.1 Definition of elasticity
13.3.2 Common properties of elasticities
13.3.3 Enzyme kinetics viewed from control analysis
13.3.4 Rates and concentrations as effects, not causes
13.4 Control coefficients
13.4.1 Definitions
13.4.2 The perturbing parameter
13.5 Properties of control coefficients
13.5.1 Summation relationships
13.5.2 Implications for large perturbations
13.5.3 Constrained enzyme concentrations
13.6 Relationships between elasticities and control coefficients
13.6.1 Connectivity properties
13.6.2 Control coefficients in a three-step pathway
13.6.3 Expression of summation and connectivity relationships in
matrix form
13.6.4 Connectivity relationship for a metabolite not involved in feedback
13.6.5 The flux control coefficient of an enzyme for the flux through its
own reaction
13.7 Response coefficients: the partitioned response
13.8 Control and regulation
13.9 Mechanisms of regulation
13.9.1 Metabolite channeling
13.9.2 Interconvertible enzyme cascades
13.9.3 The metabolic role of adenylate kinase
13.10 Computer modeling of metabolic systems
13.10.1 General considerations
13.10.2 Programs for modeling
13.10.3 The reversible Hill equation
13.10.4 Examples of computer models of metabolism
13.11 Biotechnology and drug discovery
Chapter summary
Problems
xi
319
320
323
324
327
327
327
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329
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xii
14 Fast Reactions
14.1 Limitations of steady-state measurements
14.1.1 The transient state
14.1.2 The relaxation time
14.1.3 “Slow” and “fast” steps in mechanisms
14.1.4 Ambiguities in the steady-state analysis of systems with
intermediate isomerization
14.1.5 Ill-conditioning
14.2 Product release before completion of the catalytic cycle
14.2.1 “Burst” kinetics
14.2.2 Active site titration
14.3 Experimental techniques
14.3.1 Classes of method
14.3.2 Continuous flow
14.3.3 Stopped flow
14.3.4 Quenched flow
14.3.5 Flash photolysis
14.3.6 Magnetic resonance methods
14.3.7 Relaxation methods
14.4 Transient-state kinetics
14.4.1 Systems far from equilibrium
14.4.2 Simplification of complicated mechanisms
14.4.3 Systems close to equilibrium
Chapter summary
Problems
15 Estimation of Kinetic Constants
15.1 Data analysis in an age of kits
15.2 The effect of experimental error on kinetic analysis
15.3 Least-squares fit to the Michaelis–Menten equation
15.3.1 Including error in the equation
15.3.2 Estimation of the Michaelis–Menten parameters
15.3.3 Corresponding results for a uniform standard deviation in the
rates
15.3.4 Estimating weights from replicate observations
15.4 Statistical aspects of the direct linear plot
15.4.1 Comparison between classical and distribution-free statistics
15.4.2 Application to the direct linear plot
15.4.3 Lack of need for weighting
15.4.4 Insensitivity to outliers
15.4.5 Handling of negative parameter estimates
15.5 Precision of estimated kinetic parameters
15.5.1 Experimental variance
15.5.2 Variances of the Michaelis–Menten parameters
Contents
381
381
381
382
383
385
386
388
388
390
391
391
392
393
394
396
398
399
400
400
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408
410
411
413
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424
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425
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429
429
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433
Contents
15.5.3 Standard errors
15.5.4 Km as the least precise Michaelis–Menten parameter
15.6 Generalizing the results to more than two parameters
15.6.1 The general linear model
15.6.2 Comparing models
15.7 Using existing programs for analyzing kinetic data
15.8 Residual plots and their uses
Chapter summary
Problems
xiii
434
436
438
438
439
440
443
448
449
Standards for Reporting Enzymology Data
451
Solutions and Notes to Problems
459
Index
473