STRUCTURE/FUNCTION OF EPOXIDE HYDROLASE

描述（由申请人提供）：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性化学物质（外源性物质）及其代谢物的侵害，许多环氧化物包含一组特别有害的外源性代谢物。例如，许多芳香烃和烯烃在体内容易被细胞色素P450单加氧酶氧化，形成能够烷基化核酸的环氧化物。对抗这些环氧化物的第一道化学防线是肝酶环氧化物水解酶。该酶水解环氧化物以形成相应的邻位1，2-二醇，其通常反应性较低，诱变性较低，并且由于溶解度增加而更快速地排泄。因此，环氧化物水解酶是抵抗有害化合物的第一道化学防线，这些有害化合物可以破坏遗传物质。引人注目的是，胞质或可溶性异构体的环氧化物水解酶，命名为sEH，也发挥了更大的作用以外的外源性催化剂和催化内源性脂肪酸环氧化物，如那些衍生自花生四烯酸和亚油酸的水解。这些脂肪酸环氧化物水解产生的二醇产物分别与妊娠高血压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，我们最近确定的小鼠sEH的X射线晶体结构提供了更好地理解环氧化物活化和水解反应的第一步。我们建议在sEH结构建立的基础上再接再厉。具体而言，我们的目标是：（1）通过确定位点特异性sEH变体和酶抑制剂复合物的结构来探测针对sEH提出的基于结构的机制;（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，该结构域意外地表现出金属依赖性磷酸酶活性。该领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内环氧化物水解和磷酸盐水解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.