STUDIES ON THE ACTIVE SITES AND MECHANISMS OF CYTOCHROME P450

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The cytochrome P450s are a superfamily of monooxygenases involved in the metabolism of both exogenous and endogenous compounds. Although there has been a tremendous effort to determine the details of the catalytic cycle, several aspects of these enzymes have hindered their complete characterization. These include the absence of crystal structures (for mammalian enzymes) the complexity of the catalytic cycle, presence of multiple isozymes, the diversity of substrates (for some isozymes), and the versatility of the active oxygen species. While many of the mechanisms of the actual substrate oxidation steps have been defined, the aspects of these enzymes responsible for substrate specificity and catalytic efficiency are still unknown. The goal of this research is to explore the mechanisms of oxygen activation, substrate oxidation and the topology of 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 have performed full kinetic analyses, including isotope effect experiments on wild type and mutant P450s. Stoichiometry experiments to characterize the effect of the mutations on the enzyme mechanisms are in progress.