Selective Destruction of Cytochrome P450 by Drugs

描述(由申请人提供)：拟议的研究计划解决了关于细胞色素P450酶的机制、结构、特异性和生物学作用的重要问题，这些酶在类固醇和脂肪的生物形成中发挥关键作用，药物和异物消除，药物相互作用，致癌性和毒性，以及作为生物技术的潜在工具。该研究计划的重点之一是将细菌P450酶作为结构定义的系统，以阐明与哺乳动物酶相关的细胞色素P450机制和特异性的一般特征。第二个焦点是哺乳动物的CYP4家族的脂肪酸-羟基酶，它能将花生四烯酸氧化成二十烷基类化合物，参与血管压力的控制。该计划的两个方面通过对结构和机制的潜在关注而联系在一起。我们具体建议如下：(A)进一步定义细胞色素P450酶的机制，重点是自由基反弹机制在碳氢化合物羟化中的建议作用，以及两个过渡态模型作为对反应的协同、自由基或阳离子性质相互矛盾的观察的解释的有效性；(B)探索细胞色素P450酶的结构-功能关系，集中在晶体结构可用的细菌蛋白质，特别是经历大配体依赖的活性部位构象变化的嗜热细胞色素P450，以及来自多酮类抗生素生物合成途径的两种酶P450epk和P450ervF，表现出协同作用，(C)继续开发计算机辅助方法，设计细胞色素P450抑制剂，鉴定底物，并设计具有新的特性和功能的突变体；(D)确定血红素与细胞色素P450酶中的蛋白质共价结合这一惊人发现的机制以及结构、催化和生理后果；以及(E)完善和扩展关于细胞色素P450酶的结构信息，并继续开发这些酶的同型特异性抑制物，以用于研究它们在调节血管压力和其他生理现象中的单独作用。

DESCRIPTION (provided by applicant): The proposed research program addresses important questions concerning the mechanism, structure, specificity, and biological roles of cytochrome P450 enzymes, enzymes that play critical roles in sterol and lipid biogenesis, drug and xenobiotic elimination, drug interactions, carcinogenicity and toxicity, and as potential tools in biotechnology. One focus of the research program is on bacterial P450 enzymes as structurally defined systems in which to elucidate the general features of cytochrome P450 mechanism and specificity relevant to the mammalian enzymes. The second focus is on the mammalian CYP4 family of fatty acid ?-hydroxylases that oxidize arachidonic acid to eicosanoids involved in the control of vascular pressure. The two facets of the program are linked by an underlying concern with structure and mechanism. We specifically propose the following: (a) To further define the mechanism of cytochrome P450 enzymes, with emphasis on the proposed role of the radical rebound mechanism in hydrocarbon hydroxylation and the validity of the two transition state model as an explanation for conflicting observations on the concerted, radical, or cationic nature of the reaction, (b) To explore the structure-function relationships of cytochrome P450 enzymes, concentrating on bacterial proteins for which crystal structures are available and particularly on CYPll9, a thermophilic cytochrome P450 that undergoes a large ligand-dependent active site conformational change, and P450epoK and P450ervF, two enzymes from polyketide antibiotic biosynthetic pathways that exhibit cooperativity, (c) To continue the development of computer-assisted approaches to the design of cytochrome P450 inhibitors, the identification of substrates, and the design of mutants with novel specificities and functions, (d) To determine the mechanism and the structural, catalytic, and physiological consequences of the surprising discovery that the heme is covalently bound to the protein in the CYP4 enzymes, and (e) To refine and extend the structural information on the CYP4A enzymes, and to continue the development of isoform-specific inhibitors of these enzymes to be used in examining their individual roles in the regulation of vascular pressure and other physiological phenomena.