The P450-dependent mixed-function oxidases found in almost all mammalian cells play a critical role in the metabolic activation and detoxication of many chemical carcinogens. The primary goals of the proposed research are to gain a better understanding of the structures of the active sites of several different forms of P450, to : identify the critical amino acid residues involved in the P450 active sites involved in catalysis and substrate binding and to gain a better understanding of the mechanisms by which these enzymes catalyze the reactions involved in the metabolic activation and detoxication of chemical carcinogens and other toxic agents. In order to achieve these goals the specific aims of this research proposal are: 1. To investigate mechanism-based inactivation of cytochromes P450 by 2-ethynylnaphthalene, 9-ethynylphenanthrene, and 7-ethynylcoumarin, to identify the amino acid(s) and peptide(s) at the active site modified during inactivation, and to determine the mechanism by which inactivation occurs; 2. To study the mechanism-based inactivation of cytochromes P450 by N-benzyl-1-aminobenzotriazole in order to identify the modified active site polypeptide(s) and amino acid residue(s) and to determine the mechanism of inactivation; 3. To investigate the mechanism-based inactivation of cytochromes P450 by isothiocyanates and to identify the modified amino acid residue(s) at the active site and to ascertain the catalytic step(s) blocked by the isothiocyanates; 4. To characterize the mechanism-based inactivation of cytochromes P450 by phencyclidine and 2-phenyl-2-(1-piperidynyl)propane, to identify the modified active site peptide(s) and amino acid residue(s) and the catalytic steps blocked by the inactivation; and 5. To investigate the mechanism-based inactivation of cytochromes P450 by 17-a-ethynylestradiole, to identify the modified active site polypeptide(s) and amino acid residue(s) and to determine the mechanism of inactivation. Studies aimed at investigating the effect of the various mechanism-based inactivators on the topology of the active sites of these P450s will also be investigated. Site-specific mutagenesis of active site residues modified by the : mechanism-based inactivators will also be used to determine the roles of these residues in catalysis. The identification of critical amino acid residues involved in catalysis and the determination of their specific roles in P450-catalyzed reactions will prove to be extremely valuable for developing approaches for selectively modulating the catalytic activity of these enzymes. Results of the these studies could provide information that will be extremely valuable in developing methods to decrease the risk of developing cancer.
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