P450-Mediated Dehydrogenation Mechanisms

外源性物质通过细胞色素P450氧化机制生物活化为毒性中间体是一个很好的研究方向。 认可的过程。然而，几种P450酶（例如1A 2， 2B 6、2 E1、2F 1、3A 4和4 B1）通过脱氢途径的方法， 控制选择性脱氢而不是氧化的机制尚未建立。的几 脱氢的中间体是如此活泼，以至于它们通常通过烷基化作用使P450酶变性 活性部位亲核残基。这些特异性P450酶的催化行为研究 并且它们对底物的吸附而不是吸附的倾向是非常需要的。这个假设 研究是：决定脱氢的独特的催化机制加速电子传递 某些P450酶的作用导致外源性介导的损伤和人类药物代谢的改变。的 本申请的具体目标是确定酶活性位点环境的特征， 特定细胞色素P450酶的直接脱氢机制，并确定底物结构 调节选择性脱氢而不是氧化的特征。这些目标将通过 目的：1）确定脱氢反应生成的反应中间体的结构 的原型底物，并表征酶的脱氢与氧化的偏好， 2）通过其特异性灭活剂来表征每种P450酶的灭活机制; 3） 确定控制毒物脱氢和生物活化机制的活性中心参数 4）使用共价修饰P450脱辅基蛋白的脱氢底物， 阐明指导脱氢机制或控制抑制剂/底物的关键活性位点残基 访问通道、绑定或产品发布。酶/底物对为CYP 2F 3/3-甲基吲哚， CYP 3A 4/扎鲁司特、CYP 2 E1/辣椒素和CYP 2B 6/他莫昔芬。本研究的长期目标是 阐明了细胞色素P450介导的外源性物质脱氢的机制， 产生有毒亲电中间体，以评估这些有毒中间体产生的潜在危害， 人类健康，并利用机械信息来预测脱氢和伴随的毒性和/或 酶失活（改变药物代谢），新药和异生物质。

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.