The specific aims of this proposal are to 1) explore the power and subleties of isotope effects as a tool for determining the mechanism(s) of enzymatically catalyzed reactions, and 2) to use both intramolecular and intermolecular isotope effects to study the mechanism of specific cytochrome P-450 catalyzed reactions. Numerous studies have linked the metabolic activity of these enzymes to sucg pathological conditions as kidney and liver dysfunction, teratogenesis and carcinogenesis. The ability of thes enzymes to bioactivate various substrates, e.g. polycyclic aromatic hydrocarbons, to reactive electrophilic intermediates which can covalently bind to critical biomacromolecules appears to be the basis for many of the observed toxicities. Thus an understanding of the mechanisms by which these enzymes catalyze various oxidative reactions and the factors which modulate them is crucial to the eventual understanding of the relationship between toxic liability and substrate structure. It is well known that the magnitude of an observed isotope effect in an enzymatically catalyzed reaction is in general much less than the intrinsic isotope effect for the bond breaking step. It is the later quantity however, which provides information on the transition state for the reaction. Therefore the intent of this application is to clarify how various factors like stereochemistry, branching, masking and secondary isotope effects might cause the magnitude of an observed isotope effect to deviate from the intrinsic isotope effect. Once this dependence is understood, knowledge can be gained not only on the mechanism of the specific reaction being studied but also on the nature of the reactivity of the cytochrome P-450 system. Specifically, isotope effects, isotopic labeling and the parameters outlined above will be used to explore; a) stereoselective effects associated with the omega-1 hydroxylation of octane and hexane b) the mechanism of the aromatic hydroxylation of warfarin c) the relationship between the extent of masking of an intrinsic isotope effect and the distance between two oxidatively sensitive like groupings d) the determination of the maximum intrinsic isotopr effects for the oxidation of a variety of carbon-hydrogen bonds in different electronic and steric environments e) the mechanism of N-dealkylation using substituted N-methylcarbazoles as substrates. All the studies will utilize various purified forms of cytochrome P-450 isolated from rat or the rabbit. The mmethodology for achieving the goals of the study will be quantitative mass spectrometry using stable isotopes.
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