NITROANISOLE DETOXIFICATION BY CYP2E1

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 硝基苯甲醚是一种环境污染物，其潜在毒性取决于生物过程的效率。黄嘌呤氧化酶将硝基苯甲醚活化为致癌诱变剂，而细胞色素P450，尤其是CYP 2 E1，将硝基苯甲醚氧化为易于排泄的代谢物，从而使硝基苯甲醚解毒。因此，CYP 2 E1对硝基苯甲醚的活性效率是暴露于这些污染物所造成风险的潜在重要决定因素。CYP 2 E1的动力学分析已成为评估许多小分子量化合物的活化和解毒的重要工具。与传统的酶不同，CYP 2 E1催化可能涉及多个结合位点，改变化合物的代谢。最近，我们解释了不寻常的非双曲线动力学4-硝基苯酚氧化通过效应位点的存在下，当占领，抑制反应。缺乏对这一机制的了解可能导致对污染物从体内清除的估计不正确，从而导致接触毒性。在拟议的项目中，我们将测试的假设，CYP 2 E1氧化硝基苯甲醚无毒产品的效率取决于效应位点的占用。具体而言，我们将：（1）确定硝基苯甲醚代谢的动力学机制;（2）构建CYP 2 E1与底物和效应物复合物的计算机模型，以预测非双曲线反应动力学;（3）通过光亲和标记鉴定单环分子的结合位点残基;（4）通过定点突变确认效应位点残基的功能作用。总的来说，这些发现将产生模型来解释和预测硝基苯甲醚的解毒效率，以及为未来的毒理学研究提供工具。

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.