Front Cover; Measurement and Analysis of Kinetic Isotope Effects; Copyright; Contents; Contributors; Preface; References; Chapter One: Binding Isotope Effects for Interrogating Enzyme-Substrate Interactions; 1. Introduction; 2. Equipment; 3. Materials; 3.1. Buffers; 3.2. Radiolabeled Substrates; 4. Measurement of Equilibrium BIEs Using the RED Device; 4.1. Determining Substrate Equilibration Time; 4.2. Determination of Substrate Stability; 4.3. Measurement of BIEs on Binary Complex Association; 4.4. Measurement of BIEs on Ternary Complex Association

4.5. Scintillation Counting and Spectral Deconvolution5. BIE Interpretation and Examples; 6. Conclusions; Acknowledgments; References; Chapter Two: Chemical Ligation and Isotope Labeling to Locate Dynamic Effects; 1. Introduction; 2. A General Overview of the Approach; 2.1. Production of N-Terminal Thioester Peptide EcDHFR (1-28); 2.2. Production of C-Terminal Cysteine Peptide EcDHFR-A29C (29-159); 2.3. Chemical Ligation to Form Full-Length EcDHFR; 2.4. Peptide Mass Spectrometry; 2.5. Circular Dichroism Spectroscopy; 2.6. Steady-State Kinetic Measurements

2.3. Effect of Enzyme Isotopic Labeling on Catalysis2.3.1. Effect on Steady-State Kinetic Parameters; 2.3.2. Effect on Chemical Step; 2.3.2.1. Computational Studies; 2.3.2.2. Experimental Studies: Enzyme Isotope Effects on the Rate of the Chemical Step; 2.3.2.3. Experimental Studies: Enzyme Isotope Effects on Substrate Intrinsic Kinetic Isotope Effects; 2.3.3. Regio-Specific Effects of Enzyme Isotopic Labeling on Catalysis; 3. Summary; Acknowledgments; References; Chapter Four: Extracting Kinetic Isotope Effects From a Global Analysis of Reaction Progress Curves; 1. Introduction

2. (Global) Nonlinear Fitting3. Global Fitting of a Time Course Described by Integrated Rate Equations; 4. Modeling and Fitting Time Courses Using Numerical Integration of Rate Equations; 5. Model Selection and Error Analysis; 6. Specific Examples; 6.1. DNA Polymerase; 6.2. Ethanolamine Ammonia Lyase; 6.3. Model-Free Calculation of KIEs; 7. Summary; Acknowledgments; References; Chapter Five: Natural Isotope Abundance in Metabolites: Techniques and Kinetic Isotope Effect Measurement in Plant, Anima ... ; 1. Introduction; 2. Compound-Specific 13C and 15N Analysis: History and Overview