Covalent enzyme-substrate compounds: Detection and catalytic competence

Abstract

This chapter summarizes the experimental approaches at the disposal of the enzyme chemist. The elucidation of a reaction mechanism begins with the trapping and identification of a covalent enzyme-substrate compound; the detailed examination of the catalytic mechanism requires stereochemical, kinetic, spectroscopic, and even thermodynamic data. This is obviously an ambitious undertaking, considering the fleeting nature of many intermediates. Although all enzyme reactions proceed through intermediate stages involving reversible enzyme-substrate and enzyme-substrate species, a smaller subset of enzymes forms one or more discrete covalent intermediates during their reaction cycles. Reactive intermediates facilitate catalysis (1) by tethering substrates within an active site and reducing their degrees of freedom, (2) by preserving the group transfer potential of a reactant, (3) by making a better leaving group for nucleophilic catalysis, and (4) by transferring reactants between topologically remote active sites in multifunctional enzyme complexes. Although identification of covalent enzyme-substrate compounds remains a major focus in modem enzymology, an equally significant enterprise is the demonstration of their productive participation in catalysis.