Selective Destruction of Cytochrome P450 by Drugs

描述（由申请人提供）：拟议的研究计划涉及细胞色素P450酶的机制，结构，特异性和生物学作用的重要问题，这些酶在固醇和脂质生物发生，药物和异种生物消除，药物相互作用，致癌性和毒性中起关键作用，并作为生物技术的潜在工具。研究计划的一个重点是细菌P450酶作为结构定义的系统，阐明细胞色素P450机制的一般特征和与哺乳动物酶相关的特异性。第二个重点是哺乳动物的CYP4脂肪酸家族。-羟化酶，将花生四烯酸氧化为二十烷酸，参与控制血管压力。该计划的两个方面通过对结构和机制的潜在关注联系在一起。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羟基化中的作用，以及两种过渡态模型的有效性，以解释对反应的协同、自由基或阳离子性质的矛盾观察；(b)探索细胞色素P450酶的结构-功能关系；专注于晶体结构可用的细菌蛋白质，特别是CYPll9，一种嗜热细胞色素P450，经历一个大的配体依赖的活性位点构象变化，以及P450epoK和P450ervF，两种来自聚酮类抗生素生物合成途径的酶，表现出协同性。(c)继续开发计算机辅助方法来设计细胞色素P450抑制剂，鉴定底物，以及具有新特异性和功能的突变体的设计，(d)确定血红素与CYP4酶中的蛋白质共价结合的惊人发现的机制和结构，催化和生理后果，以及(e)完善和扩展CYP4A酶的结构信息。并继续开发这些酶的异构体特异性抑制剂用于研究它们在调节血管压力和其他生理现象中的作用。

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.