Cytochrome P450 (P450) enzymes have important roles in the metabolism of carcinogens, drugs, and steroids. Although much has been learned from the available bacterial models, more remains to be understood about the principal human P450s. Major gaps exist regarding the general knowledge of rate-limiting steps in all eukaryotic P450s, which must be put in perspective to understand the influences of genetic and other variability in humans. A series of studies is proposed to characterize reactions catalyzed by human P450s 2D6, 3A4, 2A6, and 1A2, four of the major human P450s involved in drug and carcinogen metabolism and of particular significance with nitrosamines (2A6) and heterocyclic amines (1A2). These studies involve the nature of rate-limiting steps in the catalysis of model substrates. The proposed plans are based on the working central hypothesis, developed on the oasis of the literature in the field and our own experience, that rate-limiting steps for mammalian P450s differ among individual P450s and reactions but can be categorically grouped into a finite set of cases. Approaches to be used include analysis of substrate analogs, kinetic hydrogen isotope effects, pre-steady-state kinetic analysis (including the characterization of intermediates), and ligand binding. Issues regarding cooperativity will be addressed with P450s 2D6 and 3A4. Efforts to crystallize a modified form of P450 2D6 are proposed. Also, systems have been developed in this laboratory for random mutagenesis of the putative P450 """"""""substrate recognition sequences"""""""" (SRS) and other regions; these screens are highly sensitive in selection for P450s with either enhanced or attenuated catalytic activity and will be extended to further the studies on rate-limiting steps in catalysis. The basic system has already been used to obtain some human P450 1A2 mutants and will also be applied to P450 2A6. Successful completion of these collective studies should provide better understanding of the ways in which human P450s catalyze oxidation of carcinogens and drugs and the significance of variations in these enzymes.
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