Rate-limiting steps in cytochrome P-450-catalysed reactions: studies on isotope effects in the O-de-ethylation of 7-ethoxycoumarin.
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.
D015660:7-Alkoxycoumarin O-Dealkylase; D000818:Animals; D002384:Catalysis; D003374:Coumarins / Q000378:metabolism*; D006224:Cricetinae; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003903:Deuterium; D000882:Haplorhini; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D008809:Mice, Inbred C3H; D008862:Microsomes, Liver / Q000201:enzymology; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010105:Oxygenases / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D013696:Temperature
D003374:Coumarins; C017299:7-ethoxycoumarin; D003577:Cytochrome P-450 Enzyme System; D003903:Deuterium; D010105:Oxygenases; D015660:7-Alkoxycoumarin O-Dealkylase; D009251:NADPH-Ferrihemoprotein Reductase