Determination of dissociation constants and specific rate constants of enzyme-substrate (or protein-ligand) interactions from rapid reaction kinetic data

Abstract

The increasing availability of instruments capable of following absorbance or fluorescence changes in the millisecond time range has led in recent years to a large number of studies of ligandprotein interactions. Of particular interest to the enzymologist are cases where partial reactions in the catalytic cycle may be followed. For example, the reaction of a dehydrogenase with reduced pyridine nucleotide as well as the reaction of the enzymecoenzyme complex with the second substrate involved in the catalytic reaction may be followed in favorable cases by absorbance or fluorimetric changes. Numerous examples of such studies are discussed in the recent monograph by Gutfreund (1). Equally suited to such studies are enzymes utilizing pyridoxal phosphate as coenzyme, where large speetrophotometric changes are associated with the Schiff base intermediates involved in the reaction mechanisms (2). The flavoproteins, with extensive spectroscopic changes associated with the different oxidationreduction states of the flavin, as well as with enzyme-substrate intermediates, also constitute a group where much useful information has been obtained from stopped flow studies (see for example, Ref. 3). In experiments of this kind it is frequently observed that the product of the reaction appears with accurate first order kinetics