STUCTURE FUNCTION AND REGULATION OF CYTOCHROME P450 ENZYMES AND EPOXIDE HYDROLASE

这一建议是我们正在进行的研究的延伸， NADPH-细胞色素P-450结构、功能和调节 氧化还原酶、细胞色素P-450和环氧化物水解酶。 的 应用程序分为三个主要部分，每个部分 上面列出的酶。 第一个涉及（1）基础调节， 氧化还原酶基因;（2）苯巴比妥的作用机制 转录激活，（3）反式芪的分子基础 氧化物诱导;（4）结构-功能关系 氧化还原酶 氧化还原酶基因的启动子区 因为它缺乏TATA和CCAAT序列 基序，而是包含多个Sp-1的共有序列。 在 在这方面，它不同于其他基因编码的成员， 混合功能氧化酶系统以及UDP-葡萄糖醛酸基转移酶 和谷胱甘肽S-转移酶。 我们的目标是识别和 描述这些因素和序列的调节重要的 氧化还原酶基因，并阐明生物化学途径 用于异生素诱导。 此外，在没有 氧化还原酶蛋白质的晶体结构，我们将使用 计算机建模和定点突变来分析 酶的结构-功能关系。 我们的近期目标是 来鉴定对生物学功能重要的氨基酸， 活动 五个领域是表征的目标，包括 黄素（FAD和FMN），细胞色素P-450， NADPH和膜整合所需的氨基末端锚。 提案的第二部分审查了 环氧化物水解酶基因沿着的生化事件， 糖皮质激素受体依赖性地塞米松抑制 基因 下调似乎与近端AP-1位点有关， 可能涉及糖皮质激素受体对AP-1活性的抑制。 各种化合物（苯巴比妥，反式二苯乙烯） 氧化物、2-乙酰氨基芴和二乙基亚硝胺）诱导环氧化物 还将使用启动子缺失突变体研究水解酶。 它将 特别感兴趣的是比较苯巴比妥的机制， 对缺乏（氧化还原酶）和拥有（环氧化物）基因的诱导 水解酶和P-450 PCN-3）TATA和CCAAT调节序列。 在 此外，环氧化物水解酶的活性位点将被表征 使用基团特异性化学修饰 随后对受保护的 氨基酸以建立生物相关性。 本申请的第三部分提出了关于基因的问题 类固醇诱导的P-450（P-450 III）的组织和调节 基因家族）。 重点将放在理解的基础上， P-450 PCN 2和3基因的差异调节。

This proposal is an extension of our ongoing studies dealing with the structure, function, and regulation of NADPH-cytochrome P-450 oxidoreductase, cytochrome P-450, and epoxide hydrolase. The application is divided into three major parts, one for each of the enzymes listed above. The first deals with (1) the basal regulation of the oxidoreductase gene, (2) the mechanism of phenobarbital transcriptional activation, (3) the molecular basis for trans-stilbene oxide induction, and (4) the structure-function relationship of the oxidoreductase enzyme. The promoter region of the oxidoreductase gene is of particular interest since it lacks both TATA and CCAAT sequence motifs but instead contains multiple consensus sequences for Sp-1. In this respect, it differs from other genes encoding for members of the mixed-function oxidase system as well as the UDP-glucuronosyltransferase and the glutathione S-transferases. Our goal is to identify and characterize those factors and sequences important in the regulation of the oxidoreductase gene and also to elucidate the biochemical pathways for xenobiotic induction. Furthermore, in the absence of the crystallographic structure of the oxidoreductase protein, we will use computer modeling and site-directed mutagenesis to analyze the structure-function relationships of the enzyme. Our immediate goal is to identify amino acids that are functionally important for biological activity. Five domains are targeted for characterization and include the binding sites for the flavins (FAD and FMN), cytochrome P-450, NADPH, and the amino terminal anchor required for membrane integration. The second part of the proposal examines the basal regulation of the epoxide hydrolase gene along with the biochemical events responsible for the glucocorticoid receptor-dependent dexamethasone repression of the gene. Down-regulation appears to be linked to a proximal AP-1 site and may involve inhibition of AP-1 activity by the glucocorticoid receptor. The mechanisms by which various compounds (phenobarbital, trans-stilbene oxide, 2-acetylaminofluorene, and diethylnitrosamine) induce epoxide hydrolase also will be studied using promoter deletion mutants. It will be of particular interest to compare the mechanism of phenobarbital induction for genes lacking (oxidoreductase) and possessing (epoxide hydrolase and P-450 PCN-3) TATA and CCAAT regulatory sequences. In addition, the active site of epoxide hydrolase will be characterized using group specific chemical modifications in the presence and absence of an inhibitor followed by site-directed mutagenesis of the protected amino acid to establish biological relevancy. The third section of this application poses questions about the gene organization and regulation of the steroid-inducible P-450s (P-450III gene family). Emphasis will be placed on understanding the basis for the differential regulation of the P-450 PCN 2 and 3 genes.