Equilibrium Kinetic Studies of Enzyme Mechanism and Control

Abstract

This chapter discusses equilibrium kinetic studies of enzyme mechanism and control. It focuses on some aspects of the isotopic exchange kinetics at equilibrium and its evolution from the study of enzymic catalysis by the manipulation of reactants to the study of the control of catalysis by ligands that are not, themselves, catalytically transformed, inhibitors and activators, particularly of allosteric enzymes. The chapter presents particular areas of usefulness of these measurements with some examples. An important theroretical use of isotopic exchange at equilibrium was suggested to be the elucidation of compulsory pathways or binding orders. The usefulness of isotopic exchange kinetics for elucidation of compulsory binding orders was first established for the bovine heart and rabbit muscle lactate dehydrogenases. These enzymes were believed to have a compulsory pathway on the basis of binding and kinetic data. Subsequently, a compulsory pathway was demonstrated for porcine and bovine heart malate dehydrogenases and bovine liver glutamate dehydrogenase at pH 8.8. The study of enzyme catalysis at equilibrium by isotopic exchange of reactants is useful for elucidation of aspects of enzyme mechanism which may not be readily obtainable by other means. These include elucidation of a compulsory binding order, indications of fast and slow dissociation steps and whether chemical transformation is rate limiting; determination of the possibility of abortive complex formation, and, importantly, the observation of non-rate-limiting steps in catalysis. Another important aspect of equilibrium kinetics is its extension to the study of the mechanism of modifiers of enzymic catalysis, particularly with respect to effectors of allosteric enzymes.