Chapter 6 - Initial-Rate Kinetics of Multi-Substrate Enzyme-Catalyzed Reactions

Abstract

In enzyme kinetics, the terms two-substrate and three-substrate mechanisms refer to those for which the reaction rate v depends on the concentrations of two and three substrates, respectively. Most enzymes actually catalyze reactions involving two or more substrates, and, with the obvious exception of isomerases, many “one-substrate” enzymes actually have two substrates. Tightly bound cofactors that fail to dissociate after each catalytic cycle or are released only after multiple catalytic cycles are likewise kinetically indiscernible as a second substrate. This chapter focuses on the initial-rate behavior of multisubstrate enzyme-catalyzed reactions. Most enzymes utilize two or more substrates, especially those reactions involving reversibly bound coenzymes. Given the number and variety of two- and three-substrate kinetic mechanisms, experiments exclusively based on initial-rate measurements are frequently ambiguous. For this reason, enzyme kineticists must rely on other approaches, including the use of reversible competitive inhibitors, alternative substrates, isotope exchange rate measurements at or away from thermodynamic equilibrium, as well as analysis of the stereochemical course of enzyme-catalyzed reactions. The order of substrate binding determines the kinetic properties of multisubstrate reactions to such an extent that, to investigate other important aspects of catalysis, the substrate binding order must first be determined, along with their respective composite kinetic parameters Km, Vm, and Vm/Km. Compulsorily ordered kinetic mechanisms suggest the likely involvement of substrate-induced conformational changes. The kinetic mechanism for substrate addition and product release also greatly influences the way in which reaction products inhibit a given enzyme.