This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Nitroanisoles are environmental pollutants whose toxic potential depends on the efficiency of biological processes. While xanthine oxidase activates nitroanisoles to carcinogenic mutagens, cytochrome P450s, most notably CYP2E1, oxidize nitroanisoles to readily excreted metabolites, thereby committing nitroanisoles to detoxification. Thus, the efficiency of CYP2E1 activity toward nitroanisoles is a potentially important determinant for risk posed by exposure to those pollutants. Kinetic profiling for CYP2E1 has been an important tool to assess the activation and detoxification of many small molecular weight compounds. Unlike traditional enzymes, CYP2E1 catalysis may involve multiple binding sites that alter the metabolism of compounds. Recently, we explained unusual non-hyperbolic kinetics for 4-nitrophenol oxidation through the presence of an effector site, which when occupied, suppressed the reaction. Lack of knowledge of this mechanism could lead to incorrect estimates for the clearance of pollutants from the body, hence toxicity from exposure. In the proposed project, we will test the hypothesis that the efficiency of CYP2E1 oxidation of nitroanisoles to nontoxic products depends on the occupancy of an effector site. Specifically, we will: (1) determine the kinetic mechanisms for nitroanisole metabolism;(2) construct computer models for CYP2E1 complexes with substrates and effectors to predict non-hyperbolic reaction kinetics;(3) identify binding site residues for monocyclic molecules through photoaffinity labeling;and (4) confirm the functional role for effector site residues through site-directed mutagenesis. Collectively, these findings will generate models to interpret and predict the efficiency of detoxification of nitroanisoles as well as provide tools for future toxicological studies.
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