Selective Destruction of Cytochrome P450 by Drugs
Location(s): United States
The proposed research program addresses important questions concerning the mechanism, structure, specificity, and biological roles of cytochrome P450 enzymes, enzymes that play critical roles in sterol and lipid biogenesis, drug and xenobiotic elimination, drug interactions, carcinogenicity and toxicity, and as potential tools in biotechnology. One focus of the research program is on bacterial P450 enzymes as structurally defined systems in which to elucidate the general features of cytochrome P450 mechanism and specificity relevant to the mammalian enzymes. The second focus is on the mammalian CYP4 family of fatty acid m-hydroxylases that oxidize arachidonic acid to eicosanoids involved in the control of vascular pressure. The two facets of the program are linked by an underlying concern with structure and mechanism. We specifically propose the following: (a) To further define the mechanism of cytoctuome P450 enzymes, with emphasis on the proposed role of the radical rebound mechanism in hydrocarbon hydroxylation and the validity of the two transition state model as an explanation for conflicting observations on the concerted, radical, or cationic nature of the reaction, (b) To explore the structure-function relationships of cytochrome P450 enzymes, concentrating on bacterial proteins for which crystal structures are available and particularly on CYPll9, a thermophilic cytochrome P450 that undergoes a large ligand-dependent active site conformational change, and P450eDoK and P450ervF, tWO enzymes from polyketide antibiotic biosynthetic pathways that exhibit cooperativity, (c)To Continue the development of computer-assisted approaches to the design of cytochrome P450 inhibitors, the identification of substrates, and the design of mutants with novel specificities and functions, (d) To determine the mechanism and the structural, catalytic, and physiological consequences of the surprising discovery that the heme is covalently bound to the protein in the CYP4 enzymes, and (e) To refine and extend the structural information on the CYP4A enzymes, and to continue the development of isoform-specific inhibitors of these enzymes to be used in examining their individual roles in the regulation of vascular pressure and other physiological phenomena.