HYDROLYTIC ENZYMES IN THE METABOLISM OF TOXINS

评估接触外国化合物的风险取决于 关于异生代谢的知识。该提案针对的是两种酶 类，环氧化物水解酶(EHS)和酯酶，这两类都是 α/β-水解酶折叠家族。啮齿动物和人类的EHS和酯酶 在杆状病毒系统中表达的是用来发展对 基于包括X射线在内的各种工具的催化机理的研究 结晶学、同源模型、酶动力学、酶的合成 嵌合体和定点突变。一个直接的好处是 在漫长的时间里筛选人类水解酶可能的抑制剂的能力 学期目标是发展一种机械基础来预测底物和 抑制剂结合。 剧毒的环氧化物在一定程度上被EHS分解。结构性的 目标1中获得的信息有助于理解酶催化 以及几种化合物的抑制作用，包括商品除草剂和 杀虫剂。信息和基因被用来确定基础 哺乳动物细胞中sEH独特的双重定位。客观化 II解决了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的代谢谱 酸性环氧化物。一种假设认为生物活动归因于 亚油酸的环氧化物实际上是由于有毒的二元醇的产生。 将对sEH的代谢物进行检测。环氧化物的分析方法， 血清和尿液中的二醇和偶联物是基于离子的开发 TRAP-GLC-MS和免疫分析。这些氧化产物的毒性和代谢 在表达KEY的转基因细胞中检测到脂类(氧合脂) 靶细胞，如肺泡上皮和耐药细胞中的酶 例如肝细胞。这些研究将在活体内扩展以评估 外源物质与氧脂毒性的相互作用。最后，假设 外源生物改变接触工人体内的氧脂配给 通过监测高血压尿液中的氧磷脂和除草剂抑制剂进行暴露 将会受到考验。 抑制酯酶是毒性的一个公认的原因。这个 主要负责杀虫剂水解酶的酯酶 氯氰菊酯和马拉硫磷正在被提纯、克隆并在 目的III.重组酶将用于开发一种 预测这些酶的抑制剂的结构基础，从而 存在中毒风险的杀虫剂组合。对于EHS和 酯化--两步反应机制中催化四分体的假说 将会受到考验。

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.