STRUCTURE AND FUNCTION OF MAMMALIAN CYTOCHROMES P450

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cytochromes P450 perform the first step in eliminating a wide range of xenobiotics, including drugs, environmental contaminants, and procarcinogens. P450s from families 1, 2, and 3 act on distinct yet overlapping sets of diverse substrates. The molecular interactions giving rise to differential metabolism are largely undefined but are a vital prerequisite for predicting drug metabolism in vivo. Our long-term goal is to generate the structural data required to enable prediction of P450-ligand interactions that can be used in a predictive manner. The goal of this proposal is to elucidate the structural basis for the differing but overlapping substrate specificities of human 2A and 2E P450s. The hypothesis is that since both common and distinct substrates are metabolized, only a few specific interactions distinguish the metabolic capabilities of these two P450 subfamilies. Identifying those interactions is a first step toward predictive metabolism of important drug candidates and environmental xenobiotics. First, active site residues orienting marker substrates in 2A and 2E P450s will be identified. A combination of site-directed mutagenesis and analysis with common and differential substrates will be used to identify interactions responsible for distinct vs. overlapping metabolism. Second, we propose to generate a molecular structure of cytochrome P450 2E1 for comparison with 2A structures. 2E1 will be modified to increase solubility and characterized for its aggregation state, stability, and function. Promising candidate(s) will be the subject of crystallization trials with the intent to determine a high-resolution x-ray structure. Comparison of ligand interactions both between the 2A and 2E enzymes and with P450s from other subfamilies is expected to yield substantial insights into P450 structure-function diversity. This work will significantly enhance our ability to elucidate mechanisms of differential metabolism and inhibition between human P450s with potential impact on drug design, incidence of adverse drug-drug interactions, and prevention of and intervention in human carcinoma.