HYDROLYTIC ENZYMES IN THE METABOLISM OF TOXINS

外来化合物暴露风险的评估取决于 异生物质代谢的知识。该提案针对两种酶 类，环氧化物水解酶（EH）和酯酶，它们都是 α/β-水解酶折叠家族。啮齿动物和人的EH和酯酶 在杆状病毒系统中表达的基因被用来理解 基于各种工具，包括X射线， 晶体学，同源模建，酶动力学，酶合成 嵌合体和定点诱变。直接的好处是 筛选人类水解酶可能的抑制剂的能力， 长期目标是发展预测底物和 抑制剂结合。 高毒性的环氧化物部分被EH水解。结构性 目标1中获得的信息促进了对酶催化的理解 和几种化合物的抑制作用，包括商业除草剂， 杀虫剂信息和基因被用来确定 sEH在哺乳动物细胞中独特的双重定位。目的 第二部分讨论了EH活动变化影响健康的假设 通过改变内源性生物活性脂肪酸的代谢特征， 酸性环氧化物。假设生物活性归因于 亚油酸的环氧化物实际上是由于产生一种有毒的二醇 将检测sEH的代谢产物。环氧化物的分析方法， 血清和尿液中的二醇和缀合物正在基于离子 捕集器GLC-MS和免疫测定。毒性和代谢这些氧化 脂质（氧化脂质）在转基因细胞中被检测， 酶，在靶细胞如肺泡上皮细胞和抗性细胞中 例如肝细胞。这些研究将在体内进行扩展，以评估 异生物质与氧脂素毒性相互作用。最后，假设 暴露工人的氧脂素定量被外源性物质改变， 通过监测EH尿液中的氧脂素和除草剂抑制剂暴露 会得到考验 酯酶的抑制是一个公认的毒性原因。的 酯酶主要负责杀虫剂的水解 氯菊酯和马拉硫磷正在纯化、克隆和表达， 目标三重组酶将用于开发 预测这些酶的抑制剂的结构基础， 存在毒性风险的杀虫剂组合。对于EH和 酯酶在两步机制中催化四分体的假设 会得到考验

Evaluation of the risk of foreign compound exposure depends upon a knowledge of xenobiotic metabolism. This proposal targets two enzyme classes, epoxide hydrolases (EHs) and esterases, which are both members of the alpha/beta-hydrolase fold family. Rodent and human EHs and esterases expressed in the baculovirus system are used to develop an understanding of their catalytic mechanism based on a variety of tools including X-ray crystallography, homology modeling, enzyme kinetics, synthesis of enzyme chimera, and site directed mutagenesis. An immediate benefit is the ability to screen human hydrolases for possible inhibitors while a long term goal is development of a mechanistic basis to predict substrate and inhibitor binding. Highly toxic epoxides are hydrolyzed in part by EHs. The structural information gained in objective 1 advances understanding enzyme catalysis and inhibition by several compounds including commercial herbicides and insecticides. The message and gene are being used to determine the basis of the unique dual localization of the sEH in mammalian cells. Objective II addresses the hypothesis that changes in EH activity influence health by changing the metabolic profile of endogenous, biologically active fatty acid epoxides. The hypothesis that biologically activity attributed to epoxides of linoleic acid actually is due to generation of a toxic diol metabolite by the sEH will be tested. Analytic methods for the epoxides, diols and conjugates in serum and urine are being developed based on ion trap GLC-MS and immunoassay. Toxicity and metabolism of these oxidized lipids (oxylipins) are examined in transgenic cells expressing key enzymes, in target cells such as alveolar epithelium and resistant cells such as hepatocytes. These studies will be extended in vivo for evaluating interaction of xenobiotics with oxylipin toxicity. Finally, the hypothesis that oxylipin rations in exposed workers are altered by xenobiotic exposure by monitoring oxylipins and herbicide inhibitors of EH urines will be tested. Inhibition of esterases is a well established cause of toxicity. The esterases primarily responsible for the hydrolysis of the insecticides permethrin and malathion are being purified, cloned and expressed in objective III. The recombinant enzymes will be used for developing a structural basis for predicting inhibitors of these enzymes and thus combinations of insecticides which present toxic risk. For both EHs and esterases the hypothesis of a catalytic tetrad in a two step mechanism will be tested.