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. 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. Nitroanisoles are members of class of potent toxic and carcinogenic compounds, presenting a considerable danger to the human population. These pollutants are widely distributed in workplaces, especially dye industries, and the environment due to emissions from diesel and gasoline engines, ambient air particulate matter, and toxic spills. Significant efforts have identified products and the corresponding P450s responsible for nitroanisole detoxification;however, the kinetic profiles that describe the flux of these compounds to inactive metabolites require investigation. We will generate computational models of liganded complexes through two different docking approaches to identify the stoichiometry complexes and corresponding contact residues for bound ligands. These models, combined with biophysical and biochemical data, will allow us to determine the structure-function relationships for CYP2E1 relevant to the detoxification of carcinogenic nitroanisoles.
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