STRUCTURE/FUNCTION OF EPOXIDE HYDROLASE

描述(申请人提供)：随着时间的推移，无数的分解代谢途径已经进化，以保护所有生命形式免受有害的外源化学物质(外源化合物)及其代谢物的伤害，许多环氧化物包括一组特别危险的外源代谢物。例如，许多芳香烃和烯烃在体内很容易被细胞色素P450单加氧酶氧化形成能够烷化核酸的环氧化物。对这种环氧化物的第一道化学防线是肝酶环氧化物水解酶。这种酶将环氧化物水解，形成相应的邻位1，2-二醇，这些二醇通常活性较低，致突变性较小，由于溶解度增加，排泄速度更快。因此，环氧化物水解酶是抵御有害化合物破坏遗传物质的第一道化学防线。值得注意的是，环氧化物水解酶的胞液或可溶异构体，称为sEH，也发挥着比外源分解代谢更大的作用，并催化内源脂肪酸环氧化物的水解，如花生四烯酸和亚油酸衍生的环氧化物。这些脂肪酸环氧化物的二醇产物分别与妊娠高血压综合征和急性呼吸窘迫综合征有关。因此，sEH是药物设计的潜在靶点。 生物学中环氧化物活化的结构和化学基础知之甚少，我们最近测定的小鼠sEH的X射线晶体结构为更好地理解环氧化物活化和水解反应提供了第一步。我们建议在sEH结构建立的基础上再接再厉。具体地说，我们的目标是：(1)通过确定特定位点的sEH变异体和酶抑制物复合体的结构来探索sEH的基于结构的机制；(2)确定人sEH的晶体结构；(3)探索sEH的N-末端结构域的结构-机制关系，它出人意料地表现出金属依赖的磷酸酶活性。这个结构域的结构化学和生物学将帮助我们探索在同一大分子催化剂中环氧化物水解和磷酸水解酶活性的共价键的进化必要性。

DESCRIPTION (provided by applicant): Myriad catabolic pathways have evolved through time to defend all life forms against harmful exogenous chemicals (xenobiotics) and their metabolites, and many epoxides comprise a particularly sinister group of xenobiotic metabolites. For example, many aromatic and olefinic hydrocarbons are readily oxidized by cytochrome P450 monooxygenases in vivo to form epoxides capable of alkylating nucleic acids. The first line of chemical defense against such epoxides is the liver enzyme epoxide hydrolase. This enzyme hydrolyzes epoxides to form the corresponding vicinal 1,2-diols, which are typically less reactive, less mutagenic, and more rapidly excreted due to increased solubility. Therefore, epoxide hydrolase is the first line of chemical defense against harmful compounds that can damage the genetic material. Strikingly, the cytosolic or soluble isoform of epoxide hydrolase, designated sEH, also plays a greater role beyond that of xenobiotic catabolism and catalyzes the hydrolysis of endogenous fatty acid epoxides such as those derived from arachidonic acid and linoleic acid. The diol products resulting from hydrolysis of these fatty acid epoxides are implicated in pregnancy-induced hypertension and acute respiratory distress syndrome, respectively. Thus, sEH is a potential target for drug design. The structural and chemical basis of epoxide activation in biology is little understood, and our recently-determined X-ray crystal structure of murine sEH provides the first step toward a greater understanding of the epoxide activation and hydrolysis reaction. We propose to build upon the foundation established with the sEH structure. Specifically, we aim: (1) to probe the structure-based mechanism proposed for sEH by determining structures of site-specific sEH variants and enzyme-inhibitor complexes; (2) to determine the crystal structure of human sEH; (3) to explore structure-mechanism relationships for the N-terminal domain of sEH, which unexpectedly exhibits a metal-dependent phosphatase activity. The structural chemistry and biology of this domain will help us explore the evolutionary imperative for the covalent linkage of the activities of epoxide hydrolysis and phosphate hydrolysis within the same macromolecular catalyst.