P450-Mediated Dehydrogenation Mechanisms

外源生物通过细胞色素P450氧化机制对有毒中间体的生物活化是一个很好的研究方向 公认的流程。然而，几种P450酶(例如1A2， 2B6、2E1、2F1、3A4和4B1)通过脱氢途径，直到最近才被研究，并且 管理选择性脱氢而不是氧合的机制尚未建立。其中的几个 脱氢中间体非常活跃，通常通过烷基化使P450酶失活。 活性部位的亲核残基。关于这些特定P450酶催化行为的研究 而它们脱氢而不是氧化底物的倾向是至关重要的。这是一个假设 研究内容是：决定脱氢反应的独特催化机理(S)引发的电子传递 通过某些P450酶导致异物介导的损伤和改变药物在人类的代谢。这个 本申请的具体目标是确定酶活性部位环境的特征 特定细胞色素P450酶的直接脱氢机理，并确定底物结构 调节选择性脱氢而不是氧合的特性。这些目标将通过 目的如下：1)确定脱氢产生的活性中间体的结构 原型底物，并表征脱氢与氧化的酶偏好 底物；2)表征每个P450酶被其特定的失活剂灭活的机理；3) 确定控制毒物脱氢和生物活化机制的活性部位参数 通过P450酶；以及4)使用共价修饰P450脱辅基蛋白的脱氢底物来 阐明指导脱氢机制或控制抑制剂/底物的关键活性中心残基 访问渠道、绑定或产品发布。酶/底物对为CYP2F3/3-甲基吲哚， 细胞色素P3A4/扎鲁司特、细胞色素P450-EL/辣椒素和细胞色素P450-B6/他莫昔芬。这项研究的长期目标是 阐明细胞色素P450介导的外源物质脱氢过程的机制 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以及伴随的毒性和/或 新药和外源生物的酶失活(改变药物代谢)。

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.