The long-term objective of this proposal is to elucidate the structural basis for the unique functional properties of cytochromes P450 3A. These enzymes are very versatile catalysts and play a crucial role in the hepatic metabolism of a wide variety of compounds of pharmacological and toxicological interest. P450 3A enzymes are induced or inhibited by numerous foreign compounds and are involved in important drug-drug interactions in humans. In general, the functions of cytochromes P450 3A are conserved within and across species but are distinct from those of P450s from other subfamilies. Most P450 3A enzymes catalyze steroid 6 beta-hydroxylation and macrolide antibiotic metabolism and exhibit stimulation by alpha-naphthoflavone (alpha-NF). Cytochromes P450 3A accommodate some of the largest substrates known for any P450, such as cyclosporin A, and are thought to possess multiple binding sites. However, little information is available, on the structural features of the enzymes that confer their catalytic properties. Building on extensive site-directed mutagenesis and molecular modeling studies from this laboratory on determinants of P450 2B specificity, the current proposal focuses on human P450 3A4 and 3A5. 3A4 is the most highly expressed P450 in the liver of most humans and appears to metabolize more clinically important drugs than any other human P450. P450 3A4 also metabolizes the environmental contaminants benzo(a)pyrene and aflatoxin B1. P450 3A5 is expressed in the liver of approximately one in four individuals. The central hypothesis is that cytochromes P450 3A have structurally distinct substrate binding and effector sites.
The Specific Aims are to: 1) Probe the role of the substrate recognition sites identified in P450 family 2 enzymes in governing the substrate specificity and stimulation by alpha-NF of human P450 3A4 and 3A5; 2) Use random mutagenesis in conjunction with functional screening to identify residues responsible for alpha-NF stimulation of P450 3A4, and probe the biochemical basis of altered flavonoid responsiveness of key cassette, site-directed, and random mutants; 3) Localize the substrate binding and effector sites in 3-D homology models of P450 3A4 and 3A5. Delineation of the structural determinants of human P450 3A activity should aid in predicting drug-drug interactions and lead to improved drug therapy.
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