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 |
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| Authors: | G T Miwa N Harada A Y Lu |
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| Institution: | 1. Claremont McKenna, Pitzer, and Scripps Colleges, Claremont, CA, USA;2. Department of Ambulatory and Community Medicine, University of Lausanne, Lausanne, Switzerland;3. Institute of Primary Health Care (BIHAM), University of Bern, Bern, Switzerland;4. Department of Internal Medicine, Internal Medicine, Lausanne University Hospital, Lausanne, Switzerland;1. Department of Chemical Engineering, Chungnam National University, 220 Gung-Dong, Yuseong-Gu, Daejeon 305-764, Republic of Korea;2. Energy Storage Research Center, Korea Institute of Science and Technology, P.O. Box 131, Cheongryang, Seoul 130-650, Republic of Korea;3. Department of Solar Photovoltaics, Institute of Biochemical Physics, Russian Academy of Sciences, Kosygin St. 4, 119334 Moscow, Russia;4. Clean Energy Research Center, Korea Institute of Science and Technology, P.O. Box 131, Cheongryang, Seoul 130-650, Republic of Korea;5. Department of Chemistry, Changwon National University, 220 Changwondaehak-ro, Uichang-gu, Changwon, Gyeongnam 641-773, Republic of Korea |
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| Abstract: | 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. |
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