Applications of Alternative Substrate Kinetics: In Vivo and In Vitro Data on Aminoglycoside Antibiotic Inactivating Enzymes

Abstract

The study of mechanisms of enzymatic reactions is composed of two lines of inquiry. The first addresses the kinetic mechanism, which describes the order of the addition of substrates and the release of products. It also defines the central complexes between substrates and the enzymes that are possible; hence it is often the first to be undertaken. The second addresses the chemical mechanism,which describes processes of bond making and breaking that occur within central complexes and which define the roles of the amino acid side chains within the active site of the enzyme. This chapter will address the former and describe a new method for their determination. The usual kinetic mechanisms for enzymes catalyzing reactions between two substrates are shown in Fig. 33.1 (1). The first, a ping-pong kinetic mechanism, consists in the formation of only binary enzyme—substrate complexes, within two independent half-reactions, separated by a chemically modified form of free enzyme. The other three mechanisms are all sequential, and the kinetic mechanism consists simply in defining whether the binding sequence leading to and from the ternary enzyme—substrate complex is ordered or random. The Theorell-Chance mechanism is a special variant of ordered, in which the reaction segment controlling the release of the final product is rate-limiting.¹