Experimental Determination of Rate Constants in Enzymatic Reactions

Abstract

Perhaps the best way to understand the kinetic mechanism of an enzymatic reaction is to be able to determine the rate constants that are associated with each step along the catalytic pathway. Such an understanding is important in defining not only the kinetic pathway but also which steps reflect the rate-limiting process and the catalytic strategy of the enzyme. We have been developing, over the past few years, techniques that allow the determination of individual rate constants by kinetic methods. Primarily, the method involves analysis of the full time course of the enzymatic reaction by computer simulation. As is shown in this chapter, this type of analysis has proved useful: We have been able to explore questions related to the size of bimolecular rate constants in the formation of binary or ternary complexes, the rate of the chemical transformation, the evaluation of isotope effects, the rates of conformational changes, the rates of the association or dissociation of an enzyme with itself or another protein to give a species with different kinectic parameters, the rate and extent of formation of abortive complexes, the nature of product inhibition, as well as many other kinetic characteristics. This chapter deals with some of these issues.