Kinetic studies on the enzyme-substrate complex formation of p-hydroxybenzoate hydroxylase by the stopped-flow method.

Abstract

The spectrophotometric and spectrofluorometric investigations of the enzyme-substrate complex formation of p-hydroxybenzoate hydroxylase was made by the stopped-flow technique. The apparent velocity of the formation of the enzyme-substrate complex (the velocity of the absorbance change in visible and UV regions, and the velocity of the quenching of the fluorescence intensity in the FAD moiety of the holoenzyme by the substrate) was rapid enough to explain the maximal overall velocity (72 sec-1) or the activated anaerobic reduction rate (kredmax= 200 sec-1). The results were consistent with a two-step mechanism involving a rapid bimolecular association of enzyme and substrate, and a slower follow-up unimolecular process.