NITROANISOLE DETOXIFICATION BY CYP2E1

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 硝基苯甲醚是一种环境污染物，其毒性潜力取决于生物过程的效率。黄嘌呤氧化酶可激活硝基苯甲醚致癌诱变剂，而细胞色素P450，尤其是细胞色素P450，可将硝基苯甲醚氧化为易排出的代谢物，从而使硝基苯甲醚解毒。因此，对硝基苯甲醚的活性效率是暴露于这些污染物所构成风险的潜在重要决定因素。CYP2E1的动力学图谱已经成为评估许多小分子化合物的活化和解毒的重要工具。与传统的酶不同，CYP2E1催化可能涉及多个结合位点，从而改变化合物的代谢。最近，我们通过效应点的存在解释了4-硝基苯酚氧化的异常非双曲线动力学，当效应点被占据时，抑制了反应。对这一机制缺乏了解可能会导致对污染物从体内清除的错误估计，从而导致暴露的毒性。在拟议的项目中，我们将检验这样一个假设，即硝基苯甲醚氧化为无毒产品的效率取决于效应部位的占有率。具体地说，我们将：(1)确定硝基苯甲醚代谢的动力学机制；(2)构建与底物和效应物的计算机模型，以预测非双曲线反应动力学；(3)通过光亲和标记确定单环分子的结合位点残基；以及(4)通过定点突变确认效应部位残基的功能作用。总而言之，这些发现将产生解释和预测硝基苯甲醚解毒效率的模型，并为未来的毒理学研究提供工具。 硝基苯甲醚是一类具有强烈毒性和致癌性的化合物，对人类构成相当大的危险。这些污染物广泛分布在工作场所，特别是染料行业，以及由于柴油和汽油发动机排放的废气、环境空气颗粒物和有毒物质泄漏造成的环境。已经做出了重大努力，确定了负责硝基苯甲醚解毒的产物和相应的P450；然而，描述这些化合物向非活性代谢物流动的动力学曲线需要研究。我们将通过两种不同的对接方法生成配体配合物的计算模型，以确定结合配体的化学计量比配合物和相应的接触残基。这些模型结合生物物理和生化数据，将使我们能够确定与致癌硝基苯甲醚解毒相关的CYP2E1的结构-功能关系。

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.