Practical Enzyme Kinetics provides a practical how-to guide for beginning students, technicians, and non-specialists for evaluating enzyme kinetics using common software packages to perform easy enzymatic analyses.
Author(s): Alejandro G. Marangoni
Edition: 1
Year: 2002
Language: English
Pages: 248
ENZYME KINETICS......Page 4
CONTENTS......Page 10
PREFACE......Page 16
1.1 Generalities......Page 20
1.2.1 Rate Equation......Page 21
1.2.4.1 Zero-Order Integrated Rate Equation......Page 23
1.2.4.2 First-Order Integrated Rate Equation......Page 24
1.2.4.3 Second-Order Integrated Rate Equation......Page 26
1.2.4.4 Third-Order Integrated Rate Equation......Page 27
1.2.4.6 Opposing Reactions......Page 28
1.2.4.7 Reaction Half-Life......Page 30
1.2.5.1 Differential Method (Initial Rate Method)......Page 31
1.2.5.2 Integral Method......Page 32
1.3.1 Theoretical Considerations......Page 33
1.3.2 Energy of Activation......Page 37
1.4 Acid–Base Chemical Catalysis......Page 39
1.5 Theory of Reaction Rates......Page 42
1.6 Complex Reaction Pathways......Page 45
1.6.1.1 Numerical Integration......Page 47
1.6.1.2 Least-Squares Minimization (Regression Analysis)......Page 48
1.6.2 Exact Analytical Solution (Non-Steady-State Approximation)......Page 58
1.6.3 Exact Analytical Solution (Steady-State Approximation)......Page 59
2 HOW DO ENZYMES WORK?......Page 60
3.1 Progress Curve and Determination of Reaction Velocity......Page 63
3.2.1 Equilibrium Model......Page 67
3.2.2 Steady-State Model......Page 68
3.2.3 Plot of v versus [S]......Page 69
3.3 General Strategy for Determination of the Catalytic Constants K(m) and V(max)......Page 71
3.4 Practical Example......Page 72
3.5 Determination of Enzyme Catalytic Parameters from the Progress Curve......Page 77
4.1 Competitive Inhibition......Page 80
4.2 Uncompetitive Inhibition......Page 81
4.3 Linear Mixed Inhibition......Page 82
4.4 Noncompetitive Inhibition......Page 83
4.5.1 Inhibition of Fumarase by Succinate......Page 84
4.5.2 Inhibition of Pancreatic Carboxypeptidase A by β-Phenylpropionate......Page 86
4.5.3 Alternative Strategies......Page 88
5 IRREVERSIBLE ENZYME INHIBITION......Page 89
5.1 Simple Irreversible Inhibition......Page 91
5.2 Simple Irreversible Inhibition in the Presence of Substrate......Page 92
5.3 Time-Dependent Simple Irreversible Inhibition......Page 94
5.4 Time-Dependent Simple Irreversible Inhibition in the Presence of Substrate......Page 95
5.5 Differentiation Between Time-Dependent and Time-Independent Inhibition......Page 97
6.1 The Model......Page 98
6.2 pH Dependence of the Catalytic Parameters......Page 101
6.3 New Method of Determining pK Values of Catalytically Relevant Functional Groups......Page 103
7 TWO-SUBSTRATE REACTIONS......Page 109
7.1 Random-Sequential Bi Bi Mechanism......Page 110
7.1.2 Constant [B]......Page 112
7.2.1 Constant [B]......Page 114
7.2.2 Constant [A]......Page 115
7.2.3 Order of Substrate Binding......Page 116
7.3 Ping-Pong Bi Bi Mechanism......Page 117
7.3.2 Constant [A]......Page 118
7.4 Differentiation Between Mechanisms......Page 119
8 MULTISITE AND COOPERATIVE ENZYMES......Page 121
8.1.1 Basic Postulates......Page 122
8.1.2 Interaction Factors......Page 124
8.1.3 Microscopic versus Macroscopic Dissociation Constants......Page 125
8.1.4 Generalization of the Model......Page 126
8.2 Concerted Transition or Symmetry Model......Page 128
8.3 Application......Page 133
8.4 Reality Check......Page 134
9.1 Batch Reactors......Page 135
9.2 Plug-Flow Reactors......Page 137
9.3 Continuous-Stirred Reactors......Page 138
10 INTERFACIAL ENZYMES......Page 140
10.1.1 Interfacial Binding......Page 141
10.1.2 Interfacial Catalysis......Page 142
10.2 Determination of Interfacial Area per Unit Volume......Page 144
10.3 Determination of Saturation Interfacial Enzyme Coverage......Page 146
11 TRANSIENT PHASES OF ENZYMATIC REACTIONS......Page 148
11.1 Rapid Reaction Techniques......Page 149
11.2 Reaction Mechanisms......Page 151
11.2.1 Early Stages of the Reaction......Page 153
11.3 Relaxation Techniques......Page 154
12.1.1 The Model......Page 159
12.1.2 Half-Life......Page 161
12.1.3 Decimal Reduction Time......Page 162
12.1.4 Energy of Activation......Page 163
12.1.5 Z Value......Page 164
12.2 Thermodynamic Treatment......Page 165
12.3 Example......Page 169
12.3.1 Thermodynamic Characterization of Stability......Page 170
12.3.2 Kinetic Characterization of Stability......Page 175
13 MECHANISM-BASED INHIBITION......Page 177
13.1 Alternate Substrate Inhibition......Page 178
13.2 Suicide Inhibition......Page 182
13.3.1 Alternative Substrate Inhibition......Page 188
13.3.2 Suicide Inhibition......Page 189
14 PUTTING KINETIC PRINCIPLES INTO PRACTICE......Page 193
14.1 Were Initial Velocities Measured?......Page 194
14.2 Does the Michaelis–Menten Model Fit?......Page 196
14.3 What Does the Original [S] versus Velocity Plot Look Like?......Page 198
14.4 Was the Appropriate [S] Range Used?......Page 200
14.5 Is There Consistency Working Within the Context of a Kinetic Model?......Page 203
14.6 Conclusions......Page 210
15.1 Are Proteins Expressed Using Various Microbial Systems Similar to the Native Proteins?......Page 212
15.2 What Is the Mechanism of Conversion of a Zymogen to an Active Enzyme?......Page 214
15.3 What Role Does the Prosegment Play in the Activation and Structure–Function of the Active Enzyme?......Page 217
15.4 What Role Do Specific Structures and/or Residues Play in the Structure–Function of Enzymes?......Page 221
15.5.1 Charge Distribution......Page 224
15.5.2 N-Frag Mutant......Page 227
15.5.3 Disulfide Linkages......Page 229
15.6 Conclusions......Page 231
15.7 Abbreviations Used for the Mutation Research......Page 232
Books......Page 236
Selection of Classic Papers......Page 237
INDEX......Page 240
