Kinetic isotope effects on cytochrome P-450-catalyzed oxidation reactions: full expression of the intrinsic isotope effect during the O-deethylation of 7-ethoxycoumarin by liver microsomes from 3-methylcholanthrene-induced hamsters.
The intrinsic primary deuterium isotope effect for the O-deethylation of 7-ethoxycoumarin has been estimated by the Northrop method [D. B. Northrop (1977) in Isotope Effects on Enzyme-Catalyzed Reactions (Cleland, W. W., O'Leary, M. H., and Northrop, D. B., eds.), pp. 122-152, University Park Press, Baltimore] for the microsomal cytochrome P-448 system from 3-methylcholanthrene-induced hamster livers. The intrinsic isotope effect (Dk = 5.5) was found to be equivalent to the observed deuterium isotope effect, demonstrating that the isotope effect for this reaction was fully expressed by this cytochrome P-448 system. These data unequivocally demonstrate that C-H bond cleavage is the rate-limiting step in the overall reaction catalyzed by this system. The decrease in the rate of product formation, occurring as a consequence of deuterium substitution, resulted in a reduction in the quantity of substrate metabolized but was not accompanied by the change in regiospecificity observed in previous studies with a hepatic cytochrome P-448 isozyme purified from 3-methylcholanthrene-induced rats. These data demonstrate that the catalytic site of the hamster isozyme(s) offers more constraints to 7-ethoxycoumarin reorientation than does the catalytic site of the rat liver isozyme.
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003374:Coumarins / Q000378:metabolism*; D006224:Cricetinae; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000378:metabolism*; D003903:Deuterium; D004790:Enzyme Induction / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008748:Methylcholanthrene / Q000494:pharmacology*; D008862:Microsomes, Liver / Q000201:enzymology*; D008954:Models, Biological; D010084:Oxidation-Reduction; D051381:Rats; D013379:Substrate Specificity
D003374:Coumarins; C017299:7-ethoxycoumarin; D008748:Methylcholanthrene; D003577:Cytochrome P-450 Enzyme System; D003903:Deuterium