Contemporary Enzyme Kinetics and Mechanism

Reliable Lab Solutions

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• Publisher: Academic Press
• Year: 2009
• ISBN:
  9780123847447
• Edition: 3
• Topic: Biological Sciences
• Subtopic: Biochemistry
• Index: Biological Sciences

Kinetic studies of enzyme action provide powerful insights into the underlying mechanisms of catalysis and regulation. These approaches are equally useful in examining the action of newly discovered enzymes and therapeutic agents.

Contemporary Enzyme Kinetics and Mechanism, Second Edition presents key articles from Volumes 63, 64, 87, 249, 308 and 354 of Methods in Enzymology. The chapters describe the most essential and widely applied strategies. A set of exercises and problems is included to facilitate mastery of these topics.

The book will aid the reader to design, execute, and analyze kinetic experiments on enzymes. Its emphasis on enzyme inhibition will also make it attractive to pharmacologists and pharmaceutical chemists interested in rational drug design.Of the seventeen chapters presented in this new edition, ten did not previously appear in the first edition.

• Transient kinetic approaches to enzyme mechanisms
• Designing initial rate enzyme assay
• Deriving initial velocity and isotope exchange rate equations
• Plotting and statistical methods for analyzing rate data
• Cooperativity in enzyme function
• Reversible enzyme inhibitors as mechanistic probes
• Transition-state and multisubstrate inhibitors
• Affinity labeling to probe enzyme structure and function
• Mechanism-based enzyme inactivators
• Isotope exchange methods for elucidating enzymatic catalysis
• Kinetic isotope effects in enzyme catalysis
• Site-directed mutagenesis in studies of enzyme catalysis

Chapter 1 Derivation of Initial-Velocity and Isotope-Exchange Rate Equations

Charles Y. Huang, Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA

• I. Derivation of Initial-Velocity Equations
  • A. Steady-State Treatment
  • B. The Rapid-Equilibrium Treatment
  • C. The Combined Equilibrium and Steady-State Treatment
• II. Derivation of Isotope-Exchange Rate Equations
  • A. Equations for Exchanges at Equilibrium
  • B. Derivation by the Steady-State Method
• III. Concluding Remarks
• Addeendum
• References

A rate equation for an enzymic reaction is a mathematical expression that depicts the process in terms of rate constants and reactant concentrations. It serves as a link between the experimentally observed kinetic behavior and a plausible model or mechanism. The characteristics of the rate equation permit tests to be designed to verify the mechanism. Conversely, the experimental observations provide clues to what the mechanism may be, hence, what form the rate expression shall take.

Derivation of rate equations is an integral part of the effective usage of kinetics as a tool. Novel mechanisms must be described by new equations, and familiar ones often need to be modified to account for minor deviations from the expected pattern. The mathematical manipulations involved in deriving initial-velocity or isotope-exchange rate laws are in general quite straightforward, but can be tedious. It is the purpose of this chapter, therefore, to present the currently available methods with emphasis on the more convenient ones.

I Derivation of Initial-Velocity Equations

The derivation of initial-velocity equations invariably entails certain assumptions. In fact, these assumptions are often conditions that must be fulfilled for the equations to be valid. Initial velocity is defined as the reaction rate at the early phase of enzymic catalysis during which the formation of product is linear with respect to time. This linear phase is achieved when the enzyme and substrate intermediates reach a steady state or quasi-equilibrium. Other assumptions basic to the derivation of initial rate equations are as follows:

• 1. The enzyme and the substrate form a complex.
• 2. The substrate concentration is much greater than the enzyme concentration, so that the free substrate concentration is equivalent to the total concentration. This condition further requires that the amount of product formed is small, such that the reverse reaction or product inhibition is negligible.
• 3. During the reaction, constant pH, temperature, and ionic strength are maintained.

In the past decades, it was not uncommon to hear some presumably knowledgeable persons question the value of steady-state kinetics on the ground that the conditions stated in point 2 above often do not exist in vivo. To clarify this recurring doubt, one should look at these conditions as requirements for initial rate experiments in order to obtain the desired kinetic parameters. One can ask how closely the isolated enzyme retains its in vivo catalytic characteristics. However, whether steady state is ever reached in a certain metabolic pathway is a separate issue. In fact, kinetic measurements remain the best way to study par...

Table of contents

• Cover
• Title Page
• Copyright
• Table of Contents
• Preface
• Contributors
• Chapter 1: Derivation of Initial-Velocity and Isotope-Exchange Rate Equations
• Chapter 2: Practical Considerations in the Design of Initial Velocity Enzyme Rate Assays
• Chapter 3: Techniques in Coupled Enzyme Assays
• Chapter 4: Regression Analysis, Experimental Error, and Statistical Criteria in the Design and Analysis of Experiments for Discrimination Between Rival Kinetic Models
• Chapter 5: Analysis of Enzyme Progress Curves by Nonlinear Regression
• Chapter 6: Effects of pH on Enzymes
• Chapter 7: Temperature Effects in Enzyme Kinetics
• Chapter 8: Site-Directed Mutagenesis
• Chapter 9: Cooperativity in Enzyme Function
• Chapter 10: Reversible Enzyme Inhibitors as Mechanistic Probes
• Chapter 11: Application of Affinity Labeling for Studying Structure and Function of Enzymes
• Chapter 12: Mechanism-Based Enzyme Inactivators
• Chapter 13: Transient Kinetic Approaches to Enzyme Mechanisms
• Chapter 14: Rapid Quench Kinetic Analysis of Polymerases, Adenosinetriphosphatases, and Enzyme Intermediates
• Chapter 15: Presteady-State Kinetics of Enzymatic Reactions Studied by Electrospray Mass Spectrometry with Online Rapid-Mixing Techniques
• Chapter 16: Isotope Exchange Methods for Elucidating Enzymic Catalysis
• Chapter 17: Positional Isotope Exchange as Probe of Enzyme Action
• Chapter 18: Enzymatic Transition-State Analysis and Transition-State Analogs
• Chapter 19: Determining Transition States from Kinetic Isotope Effects
• Chapter 20: Computational Methods for Transition State and Inhibitor Recognition
• Appendix: Selected Exercises and Problems
• Index