Non-Michaelian monooxygenase kinetics: studies using competitive inhibitors

Abstract

Microsomal l~onooxygenase activity has been found frequently to depart from Mic~elis-Menten kinetics in that Lineweaver-Burk plots for the oxidation of a number of xenobiotics have exhibited downwards curvature at high substrate concentrations [l-4]. These results are consistent with catalysis being effected by more than one enzyme, each with a different IS,, value, and this possibility has received some support in the finding that endop~asmic reticulum contains multiple monooxygenases [2,3,5]. Such kinetic behaviour could however be due to a single enzyme possessing multiple interacting sites ]6,7] and it has been proposed that microsom~ monooxygenase activity may be regulated by a ligand concentration-dependent mechanism exhibiting negative cooperativity [8]. Here, we have shown that Michaeiis-Lenten kinetics did not describe the kinetics of O-demethylation of p-nitroanisole by a rat liver microsomal preparation when enzyme activity was measured over a 1300fold range of substrate concentration. In addition, results of studies with 3 competitive inhibitors precluded multiple independent catalytic sites as the source of the observed non-Michaelian kinetics of ether cleavage thereby lending support to the hypothesis that negative cooperativity may be a feature of the control of microsomal monooxygenase activity.