Rate-limiting steps in cytochrome P-450-catalysed reactions: studies on isotope effects in the O-de-ethylation of 7-ethoxycoumarin.

Abstract

Primary deuterium isotope effects ranging from 2 to 6 were measured for the O-de-ethylation of 7-ethoxycoumarin catalysed by microsomal and purified cytochrome P-450 isozymes. Interpretation of the observed deuterium isotope effect in terms of the contribution of the C-H bond-cleavage step to the overall rate requires the determination of the intrinsic isotope effect. Evidence is presented that at least one irreversible step occurs before C-H bond cleavage which kinetically divides the cytochrome P-450 reaction cycle into two portions. Intermolecular primary isotope effects are shown to provide kinetic information only about steps following the irreversible step. Consequently, the isotope effect may be a measure of the rate limitation imposed by the C-H bond-cleavage step in the sequence of events following the irreversible step but be totally independent of other slow steps preceding the irreversible step. These results indicate that multiple rate-limiting steps may exist in cytochrome P-450-catalysed reactions.