STUDIES ON THE ACTIVE SITES AND MECHANISMS OF CYTOCHROME P450

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The cytochromes P450 are a family of isozymes capable of oxidizing a wide variety of both endogenous and exogenous compounds. Two characteristics of these enzymes make it possible for a limited number of isozymes to metabolize a vast and varied array of chemical compounds. The first is the generally broad substrate and regiospecificity presumably due to relatively nonspecific substrate binding characteristics and multiple binding orientations. The second is a versatile active oxygenating species that is capable of oxidizing a variety of functional groups. The goal of this research is to explore the mechanisms of oxygen activation, substrate oxidation and the topology of the P450 active sites. Methods used in the project include recombinant DNA techniques, determination of enzyme and isotope effect kinetics, and kinetic analysis of both wild type and mutant enzymes. In the past, we have derived several equations for comprehensive kinetic models to describe the observed kinetic isotope effects on cytochrome P450 catalyzed oxidations. These models suggest that the observed isotope effects can provide information on both binding conformations and the amount of uncoupled electron flux that results in water formation. Previous studies on the metabolism of testosterone by several of the expressed P450 isozymes and their chimeric and mutant forms (developed by Dr. Frank Gonzalez) revealed that modification of a few amino acid residues in critical positions can markedly affect the pattern of metabolites. We are now in the process of performing full kinetic analyses, including isotope effect experiments, and stoichiometry experiments to characterize wild-type enzymes and the effect of the mutations on the enzyme mechanisms.