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 in the P450 active sites involved in catalysis and substrate binding and to gain a better understanding of the mechanisms by which they 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: l. To investigate the mechanism-based inactivation of several purified isozymes of cytochrome P450 by 9-ethynylphenanthrene, 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 P450 by 2- ethynylnaphthalene in order to identify the modified active site polypeptide(s) and amino acid residue(s) and determine the mechanism of inactivation; 3. To determine the structure-activity relationships and enzyme specificities for mechanism-based inactivation of P45Os by a series of polycyclic aromatic acetylenes; 4. To investigate mechanism-based inactivation of P450 by N-methylcarbazole in order to identify the modified amino acid residue(s) at the active site and ascertain the catalytic step(s) blocked by N-methylcarbazole; 5. To characterize P450 inactivation by N-benzyl-l-aminobenzotriazole, to identify the modified active site peptide(s) and amino acid residue(s) and the catalytic step(s) blocked by inactivation; 6. To investigate the mechanism-based inactivation of P450 by n-propylxanthate, to identify the modified active site peptide(s) and amino acid residue(s) and determine the mechanism of inactivation; 7. To investigate the metabolism of hypersensitive radical probes and determine substrate deuterium isotope effects for hydrocarbon hydroxylations by several P45Os in order to gain a better understanding of the mechanism(s) involved in P450-catalyzed hydrocarbon hydroxylations; and 8. To use site-specific mutagenesis of P450 2B1 and 2B2 to identify amino acid residues that play a critical role in substrate binding and catalysis by P450. 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. The results of these studies could provide information for developing methods to decrease the risk of developing cancer.
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