Enzyme Kinetics: Principles and Methods

 Content: 1 Multiple Equilibria 5 --
 1.1 Diffusion 5 --
 1.2 Interaction of Ligands and Macromolecules 10 --
 1.2.1 Binding Constants 10 --
 1.2.2 Derivation of the Binding Equation 11 --
 1.3 Macromolecules with Identical Independent Binding Sites 11 --
 1.3.1 General Binding Equation 11 --
 1.3.2 Graphic Representation of the General Binding Equation 17 --
 1.3.3 Binding of Various Ligands, Competition 22 --
 1.4 Macromolecules with Non-Identical, Independent Binding Sites 26 --
 1.5 Macromolecules with Identical, Interacting Binding Sites, Cooperativity 28 --
 1.5.1 The Hill Equation 28 --
 1.5.2 The Adair Equation 30 --
 1.5.3 The Pauling Model 32 --
 1.5.4 Allosteric Enzymes 32 --
 1.5.5 The Symmetry Model 33 --
 1.5.6 The Sequential Model and Negative Cooperativity 38 --
 1.5.7 Physiological Aspects of Cooperativity 41 --
 1.5.8 Analysis of Cooperativity 44 --
 1.5.9 Examples of Allosteric Enzymes 45 --
 1.6 Non-Identical, Interacting Binding Sites 48 --
 2 Enzyme Kinetics 51 --
 2.1 Reaction Order 51 --
 2.1.1 First Order Reactions 51 --
 2.1.2 Second Order Reactions 53 --
 2.1.3 Zero Order Reactions 54 --
 2.2 Steady-State Kinetics and the Michaelis-Menten Equation 55 --
 2.2.1 Derivation of the Michaelis-Menten Equation 55 --
 2.3 Analysis of Enzyme Kinetic Data 58 --
 2.3.1 Graphical Representations of the Michaelis-Menten Equation 58 --
 2.3.2 Determination of the Reaction Rate 71 --
 2.4 Reversible Enzyme Reactions 75 --
 2.4.1 Rate Equation for Reversible Enzyme Reactions 75 --
 2.4.2 The Haldane Equation 77 --
 2.4.3 Product Inhibition 78 --
 2.5 Enzyme Inhibition 80 --
 2.5.1 Reversible Enzyme Inhibition 81 --
 2.5.2 Irreversible Enzyme Inhibition 103 --
 2.5.3 Enzyme Reactions with Two Competing Substrates 106 --
 2.6 Multi-Substrate Reactions 108 --
 2.6.2 Random Mechanism 109 --
 2.6.3 Ordered Mechanism 113 --
 2.6.4 Ping-Pong Mechanism 115 --
 2.6.5 Haldane Relationships in Multi-Substrate Reactions 117 --
 2.6.6 Mechanisms with More than Two Substrates 118 --
 2.6.7 Other Notations for Multi-Substrate Reactions 120 --
 2.7 Derivation of Rate Equations of Complex Enzyme Mechanisms 120 --
 2.7.1 King-Altman Method 120 --
 2.7.2 Simplified Derivations According to the Graph Theory 126 --
 2.7.3 Combination of Equilibrium and Steady-State Assumptions 127 --
 2.8 Kinetic Treatment of Allosteric Enzymes 129 --
 2.8.1 Hysteretic Enzymes 129 --
 2.8.2 Kinetic Cooperativity, the Slow Transition Model 130 --
 2.9 Special Enzyme Mechanisms 131 --
 2.9.1 Kinetics of Immobilised Enzymes 131 --
 2.9.2 Polymer Substrates 138 --
 2.10 pH and Temperature Dependence of Enzymes 139 --
 2.10.1 pH Optimum Curve and Determination of pK Values 139 --
 2.10.2 pH Stability of Enzymes 141 --
 2.10.3 Thermal Stability of Enzymes 142 --
 2.10.4 Temperature Dependence of Enzyme Reactions 143 --
 2.11 Isotope Exchange 146 --
 2.11.1 Isotope Exchange Kinetics 146 --
 2.11.2 Isotope Effects 150 --
 2.12 Application of Statistical Methods in Enzyme Kinetics 153 --
 2.12.1 General Remarks 153 --
 2.12.2 Statistical Terms Used in Enzyme Kinetics 156 --
 3 Methods 161 --
 3.1 Methods for the Investigation of Multiple Equilibria 161 --
 3.1.1 Equilibrium Dialysis and General Aspects of Binding Measurements 162 --
 3.1.2 Continuous Equilibrium Dialysis 168 --
 3.1.3 Ultrafiltration 170 --
 3.1.4 Gel Filtration 172 --
 3.1.5 Ultracentrifugation Methods 175 --
 3.2 Electrochemical Methods 180 --
 3.2.1 The Oxygen Electrode 181 --
 3.2.2 The CO[subscript 2] Electrode 183 --
 3.2.3 Potentiometry, Oxidation-Reduction Potentials 183 --
 3.2.4 The pH-Stat 184 --
 3.2.5 Polarography 185 --
 3.3 Calorimetry 186 --
 3.4 Spectroscopic Methods 188 --
 3.4.1 Absorption Spectroscopy 190 --
 3.4.2 Bioluminescence 201 --
 3.4.3 Fluorescence 201 --
 3.4.4 Circular Dichroism and Optical Rotation Dispersion 212 --
 3.4.5 Infrared and Raman Spectroscopy 217 --
 3.4.6 Electron Spin Resonance Spectroscopy 219 --
 3.5 Measurement of Fast Reactions 222 --
 3.5.1 Flow Methods 223 --
 3.5.2 Relaxation Methods 231 --
 3.5.3 Flash Photolysis, Pico- and Femtoseconds Spectroscopy 236 --
 3.5.4 Evaluation of Rapid Kinetic Reactions (Transient Kinetics) 238.