STRUCTURAL DETERMINANTS OF P450 1A1 AND 1A2 SPECIFICITY

West Virginia University, Morgantown WV

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Abstract

DESCRIPTION (provided by applicant): Human cytochromes P450 1A1 and 1A2 are important oxidative enzymes involved in carcinogenesis and drug metabolism. P450 1A1, found mainly in lung tissues, is thought to be linked to lung cancer since this enzyme is able to oxidize benzo[a]pyrene and other polycyclic aromatic hydrocarbons (PAHs) to mutagenic diols and epoxides. Human P450 1A2 is a typical hepatic P450. It catalyzes oxidation of heterocyclic amines to genotoxic products as well as oxidation of drugs. The elucidation of structure-function relationships of these enzymes will help in the design of effective therapeutic agents that would decrease the incidence of cancer. Although cytochromes P450 1A1 and 1A2 are very similar is sequence (72% identity), they differ in substrate and inhibitor specificities. In general, 1A1 exhibits a preference for polyaromatic hydrocarbons, while 1A2 is associated with heterocyclic amine substrates. The long-term objective of the proposed research is to elucidate the structural basis for those differences. The overall structure of these enzymes is likely to be similar, with functional differences caused by the presence of a number of key amino acid. Thus, substitution of these residues in one enzyme to mimic the other should interconvert activities. This hypothesis will be tested by molecular modeling methods, in which 1A1 and 1A2 homology models will be used to identify key residues and to analyze enzyme-substrate interactions. Modeling predictions will be followed by the construction, expression and functional evaluation of appropriate mutant proteins. Innovative features of this proposal include extensive utilization of molecular dynamics simulations and binding free energy calculations to predict substrate specificity and inhibitor selectivity. The multidisciplinary nature of this project, which combines two very different areas of investigation, such as computational chemistry and biochemistry/molecular biology, should be highly advantageous in studies involving complex biochemical and toxicological issues. The proposed research should provide important information about the molecular basis of P450 1A function and lay a groundwork for the rational design of 1A inhibitors. This, in turn, may help to design effective anticancer drugs.