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1. Introduction
1.1. Proficiency of Enzyme Catalysis
1.2 Catalytic Strategies Used by Enzymes
1.2-a. Proximity Effects
1.2-b. Desolvation
1.2-c. Electrostatic Destabilization
1.2-d. Managing Intrinsic Binding Energy
1.2-e. Orienting Reactive Groups
1.2-f. Destabilizing Reactant Ground States
1.2-g. Acid-Base Catalysis
1.2-h. Hydrogen Bonding
1.2-i. Covalent Catalysis
1.2-j. Metal Ion Effects
1.2-k. Conformational Flexibility
1.3. Chymotrypsin: A Prototypical Enzyme

2. Basic Chemical Kinetics
2.1 A Few of the Basics
2.2 Reaction Order & Molecularity
2.3 Integrated Rate Laws
2.3-a. First-Order Reactions
2.3-b. Series First-Order Reactions
2.3-c. Second-Order Reactions
2.3-d. Pseudo-Second-Order Reactions
2.3-e. Opposing First-Order Reactions
2.4 Barriers to Reaction
2.4-a. Arrhenius Theory
2.4-b. Smoluchowski Equation
2.4-c. Transition State Theory (TST)
2.5 Reaction Mechanisms
2.6 Reaction Coordinate Diagrams
2.7 Energetics of Triose-P Isomerization
2.8 Timescales of Chemical Processes
2.9 Kinetics versus Dynamics
2.10 Concluding Remarks
Problem Set

3. Initial-Rate Kinetics of One-Substrate Enzymes
3.1 Michaelis-Menten Kinetics
3.2 Briggs-Haldane Treatment
3.3 KS and Km in Michaelis and Haldane Treatments
3.4 Meaning of kcat and Vm
3.5 Meaning of kcat/Km
3.6 Two-Intermediate Rate Law
3.7 Persistence of the Steady-State Phase
3.8 Concluding Remarks
Recommended Reading
Problem Set

4. Measuring Initial Velocities of Enzyme-catalyzed Reactions
4.1 The Initial-Rate Experiment
4.1-a. Lineweaver-Burk Plot
4.2-b. [S]/v versus [S] Plot
4.2 Experimental Design
4.3 Quantifying Product Formation
4.3-a. Absorption Spectroscopy
4.3-b. Fluorescence Spectroscopy
4.3-c. Radiometric Assays
4.3-d. Polarimetry & Circular Dichroism
4.3-e. Manometry
4.3-f. pH Measurements
4.3-g. Mass Spectroscopy
4.3-h. Light Scattering
4.3-i. Turbidity
4.3-j. Calorimetry
4.4 Coupled Enzyme Assays
4.5 Statistical Analysis of Enzyme Rate Data
4.6 Concluding Remarks
Further Reading
Problem Set

5. Multi-substrate Enzyme Kinetic Mechanisms
5.1 Multi-Substrate Kinetic Mechanisms
5.2 Theorell-Chance Mechanism
5.3 Ordered Ternary Complex Mechanism
5.4 Rapid-Equilibrium Random Mechanism
5.5 Ping Pong Bi Bi Mechanism
5.6 Bisubstrate Initial-Rate Data
5.7 How Bisubstrate Enzyme Kinetic Mechanisms are Differentiated
5.7-a. Initial-Rate Experiments
5.7-b. Product Inhibition Experiments
5.7-c. Alternative Substrate Experiments
5.7-d. Competitive Inhibition Experiments
5.7-e. Isotope Exchange at Equilibrium
5.7-f. Fast Kinetic Techniques
5.8 Iso-Mechanisms
5.9 Three-Substrate Kinetic Mechanisms
5.10 Concluding Remarks
Problem Set

6. Fast Kinetic Techniques for Probing Enzyme-Catalyzed Reactions
6.1 A Fuller Picture of Enzyme Catalysis
6.2 Stopped-Flow Technique
6.3 Rapid Mix/Quench Technique
6.4 Global Statistical Analysis
6.5 Relaxation Methods
6.5-a. Temperature-Jump Technique
6.5-b. Pressure-Jump Technique
6.5-c. Flash Photolysis
6.5-d. Nuclear Magnetic Resonance
6.6 Basics of Chemical Relaxation Theory
6.7 Examples of Fast Reaction Studies
6.7-a. Aspartate Aminotransferase
6.7-b. Ribonuclease
6.7-c. Antibody-Antigen Interactions
6.8 Concluding Remarks
Further Reading
Problem Set

7. Factors Affecting Enzyme Rates
7.1 pH Effects on Enzyme Kinetics
7.1-a. pH-Rate Behavior
7.1-b. Derivation of pH Functions
7.2 Temperature Effects on Enzyme Rates
7.3 Pressure Effects on Enzyme Rates
7.4 Activator Effects on Enzyme Rates
7.4-a. Types of Activators
7.4-b. Activator Rate Equations
7.4-c. Metal Ion as Enzyme Activators
7.5 Effect of Mutation on Enzyme Kinetics
7.5-a. Enzymes as Targets for Mutation and Change
7.5-b. Alanine Scanning Mutagenesis
7.5-c. Probing Catalysis by Site-Directed Mutagenesis
7.5-d. Probing Triose-Phosphate Isomerase by Mutagenesis
7.6 Concluding Remarks

8. Kinetic Isotope Effects
8.1 Kinetic Isotope Effects
8.2 Primary Kinetic Isotope Effects
8.2-a. Basics
8.2-b. Other Factors Influencing Primary KIEs
8.2-c. Effects on Equilibria
8.2-d. Quantum Mechanical Tunneling
8.3 Secondary Kinetic Isotope Effects
8.4 Why Some Enzymatic KIEs Are Masked
8.5 Scope of KIE Measurements
Further Readings

9. Inhibitor Effects on Enzyme-Catalyzed Reactions
9.1 Reversible versus Irreversible Inhibition
9.2 Reversible Enzyme Inhibitors
9.2-a. Competitive Inhibition
9.2-b. Noncompetitive Inhibition
9.2-c. Uncompetitive Inhibition
9.2-d. Slow, tight-binding Inhibition
9.2-e. Transition-State Inhibitors
9.3 Other Types of Enzyme Inhibition
9.3-a. Product Inhibition
9.3-b. Multi-Substrate Geometric Inhibition
9.3-c. Fragment-based Inhibitor Design
9.3-d. Photoaffinity Inhibition
9.3-e. Mechanism-Based Inhibition
9.4 Concluding Remarks
Problem Set

10. Enzyme Cooperativity
10.1 Cooperativity of K-Systems and V-Systems
10.2 Specific versus Nonspecific Binding
10.3 O2 Interactions with Hemoglobin Drove the Development of Cooperativity Models
10.4 Hill Model for Cooperativity
10.5 Monod-Wyman-Changeux "Concerted Transition" Model for Cooperativity
10.6 Adair-Koshland Sequential Model
10.7 Hysteretic Enzymes
10.8 Concluding Remarks

11. Kinetics of Force-Generating Enzymes

• Provides practical information about how to work with enzymes and design experiments to identify new inhibitors or activators
• Includes detailed step-by-step derivations of rate equations, showing tried-and-true ways to confirm that the equations obtained are correct
• Arranged for use both as a desk reference (with over 200 equations and more than 400 key references) or as a textbook (with 8-10 problems/exercises at the end of each chapter)

Daniel L. Purich, Professor of Biochemistry and Molecular Biology, University of Florida Health Science Center, FL, USA.