P450-Mediated Dehydrogenation Mechanisms

P450 介导的脱氢机制

• 批准号: 7544947
• 负责人: Garold S Yost
• 金额: $ 28.16万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7544947
• 关键词: Acetylcysteine; Active Sites; Alkylation; Apoproteins; Behavior; Binding; Biochemical; CYP2B6 gene; CYP2E1 gene; CYP2F3 gene; CYP3A4 gene; Capsaicin; Characteristics; Chemicals; Cleaved cell; Complementary DNA; Cyanogen Bromide; Cytochrome P450; Deuterium; Drug Interactions; Electron Transport; Environment; Enzymes; Escherichia coli; Exhibits; Glutathione; Goals; Health; Human; Injury; Kinetics; Label; MALDI-TOF Mass Spectrometry; Mediating; Methods; Molecular; Molecular Models; Pathway interactions; Peptide Fragments; Pharmaceutical Preparations; Predisposition; Process; Production; Property; Recombinants; Research; Research Personnel; Site; Skatole; Specificity; Structure; Tamoxifen; Time; Toxic effect; Xenobiotics; analog; base; dehydrogenation; drug metabolism; enzyme substrate; human SLC25A5 protein; inhibitor/antagonist; insight; liquid chromatography mass spectrometry; molecular modeling; mutant; preference; programs; toxicant; zafirlukast

Bioactivation of xenobiotics to toxic intermediates through cytochrome P450 oxygenation mechanisms is a well recognized process. However, the production of electrophilic intermediates by several P450 enzymes (e.g. 1A2, 2B6, 2E1, 2F1, 3A4, and 4B1), through dehydrogenation pathways has only recently been investigated, and the mechanisms that govern selective dehydrogenation rather than oxygenation are not established. Several of the dehydrogenated intermediates are so reactive that they inactivate the P450 enzymes, generally through alkylation of active site nucleophilic residues. Research concerning the catalytic behavior of these specific P450 enzymes and their propensity to dehydrogenate rather than oxygenate substrates is vitally needed. The hypothesis of this research is: the unique catalytic mechanism(s) offacilitated electron transport that determines dehydrogenation by certain P450 enzymes results in xenobiotic-mediated injury and altered drug metabolism in humans. The specific goals of this application are to determine the characteristics of the enzyme active-site environment that direct dehydrogenation mechanisms of specific cytochrome P450 enzymes, and to define the substrate structural features that regulate selective dehydrogenation rather than oxygenation. These goals will be realized through the following aims: 1) To determine the structures of the reactive intermediates that are produced by dehydrogenation of prototypical substrates, and characterize enzyme preferences for dehydrogenation vs. oxygenation of the substrates; 2) To characterize the mechanisms of inactivation of each P450 enzyme by its specific inactivator; 3) To define the active-site parameters that control the mechanisms of dehydrogenation and bioactivation of toxicants by P450 enzymes; and 4) To use the dehydrogenation substrates that covalently modify the P450 apoproteins to elucidate critical active-site residues that direct the dehydrogenation mechanism, or that control inhibitor/substrate access channels, binding, or product release. The enzyme/substrate pairs are CYP2F3/3-methylindole, CYP3A4/zafirlukast, CYP2El/capsaicin, and CYP2B6/tamoxifen. The long-term goals of this research are to elucidate the mechanisms of cytochrome P450-mediated dehydrogenation of xenobiotics in processes that generate toxic electrophilic intermediates, to assess the potential harm engendered by these toxic intermediates to human health, and to utilize mechanistic information to predict dehydrogenation, and concomitant toxicities and/or enzyme inactivation (altered drug metabolism), of new drugs and xenobiotics.

通过细胞色素P450氧合机制将异生物生物学生物活化到有毒中间体是一种井 公认的过程。然而，通过几种P450酶产生亲电中间体（例如1A2， 2B6、2E1、2F1、3A4和4B1）通过脱氢途径进行了研究，直到最近才开始研究，并通过 未建立控制选择性脱氢而不是氧合的机制。几个 脱氢中间体具有反应性，以至于它们通常通过烷基化使P450酶失活 活性位点亲核残基。有关这些特定P450酶的催化行为的研究 并且它们倾向于脱氢而不是氧化底物的倾向。这个假设 研究是：确定脱氢的独特催化机制（S）非活化电子传输 通过某些P450酶会导致异种生物介导的损伤，并改变了人类的药物代谢。这 本应用的具体目标是确定酶的活动地点环境的特征 特定细胞色素P450酶的直接脱氢机制，并定义底物结构 调节选择性脱氢而不是氧合的特征。这些目标将通过 以下目的：1）确定由脱氢产生的反应性中间体的结构 原型底物的特征，表征了脱氢作用与氧合的酶偏好 底物； 2）表征每种P450酶通过其特异性灭活剂的灭活机制； 3） 定义控制有毒物质脱氢和生物活化机制的主动位点参数 由P450酶； 4）使用将P450 paprototin重价修改为的脱氢底物 阐明指导脱氢机制或控制抑制剂/底物的关键活动位点残基 访问通道，绑定或产品发布。酶/底物对是CYP2F3/3-甲基吲哚， CYP3A4/Zafirlukast，CYP2EL/CAPSAICIN和CYP2B6/TAMOXIFEN。这项研究的长期目标是 阐明细胞色素P450介导的异生元介导的过程中的机理 产生有毒的亲电中间体，以评估这些有毒中间体对 人类健康，并利用机械信息来预测脱氢和毒性和/或毒性和/或 酶失活（改变的药物代谢），新药和异种生物。