Practical Aspects of Measuring Initial Rates and Reaction Parameters
This chapter focuses on the practical design and execution of initial-rate enzyme kinetic experiments, including how the initial-velocity condition is verified, how rate measurements are actually conducted, and how the resulting data can be analyzed. This chapter also provides best-practice advice for making absorbance and fluorescence measurements, tips for handling multisubstrate reactions, recommendations for using stable and radioactive isotopes to measure reaction rate, protocols for dealing with metal nucleotide interactions, suggestions for detecting and quantifying covalently bound enzyme intermediates, and various approaches for determining equilibrium constants of enzyme-catalyzed reactions. The elucidation of catalytic and regulatory mechanisms is rooted in a deep understanding of an enzyme's properties. An assay is a tool for characterizing enzymes and for conducting informative kinetic experiments. Establishing a reliable initial-rate assay for a newly discovered enzyme also presents the opportunity to uncover new catalytic and regulatory properties. The discovery of a new enzyme-catalyzed reaction begins with the observed disappearance of the substrate or appearance of a product under well-defined reaction conditions. The identity and stability of the product(s) are determined by various analytical chemical approaches that allow determination of a substance's structure on the basis of its spectral properties and reactivity. The stoichiometry of the reaction is also determined by quantifying the moles of substrate consumed and the moles of product formed. Initial-rate kinetic experiments are then employed to reveal optimal reaction conditions, to evaluate the Michaelis constant, Km, and maximal velocity, Vm, to establish the order of substrate addition and product release, and to define the basic modes of inhibitor and activator action.