Chapter 5 - Initial-Rate Kinetics of One-Substrate Enzyme-Catalyzed Reactions

Abstract

The initial-velocity approach offers a versatile range of experimental paradigms for obtaining significant information about enzyme catalysis and control. The initial-rate approach remains a powerful kinetic approach, one that has been employed to discover the vast majority of principles of enzyme action. This chapter deals with initial-rate kinetic approaches and develops the basic theory for the kinetics of one-substrate enzyme-catalyzed reactions, beginning with the rapid-equilibrium treatment of Michaelis–Menten and the steady-state treatment of Briggs and Haldane. While rate parameters like Km, Vmax, and Vmax/Km provide valuable insights into the operation of one-substrate enzymes, initial rate analysis of enzymes provides an incomplete picture of catalysis, especially with regard to the nature of the rate-determining step(s), and the number of elementary reactions. This chapter also deals with a two-intermediate steady state case, the effect of multiple internal isomerizations on steady-state kinetics, as well as the effect of total enzyme concentration on reaction rate. Then procedures for plotting enzyme rate data are considered and the definitions and conceptual implications of Michaelis constants, turnover numbers, specificity constants, commitment to catalysis, catalytic proficiency, internal equilibria, and enzyme perfection are discussed. The chapter also covers related aspects of ribozyme kinetics, the kinetics for simultaneous enzymatic action on the same or different substrates, the kinetics of enzymatic enantiomeric enrichment, the kinetics of enzymes acting on polymeric substrates, processive versus distributive enzyme kinetics, random scission kinetics, the kinetics of substrate-assisted catalysis, the use of micro-arrays, and phage display to profile enzyme specificity, as well as the effects of nonproductive polymeric substrate binding.