MEMBRANE-BOUND DETOXICATION ENZYMES

潜在有毒外来物（异生物质）的代谢和解毒 真核生物中的分子依赖于几种膜的参与， 结合的酶如细胞色素P-450、环氧化物水解酶和UDP- 葡萄糖醛酸基转移酶参与连贯的代谢途径。 为了真正理解代谢和解毒的途径， 因此，有必要对外来生物有一个基本的了解。 每种酶的作用机制、底物特异性和结构 参与或可能参与新陈代谢的细胞。 研究 本提案中概述的计划旨在加强这种理解 对于两种膜结合解毒酶，环氧化物 水解酶（EH）和UDP-葡糖醛酸基转移酶（UDPGT）。 微粒体EH 催化水与环氧化物的反式加成， 由细胞色素P-450对不饱和烃的作用产生。 UDPGT催化葡萄糖醛酸基从UDP-葡萄糖醛酸转移 涉及带有亲核官能团的疏水分子，例如- OH、-SH、-NH2和-COOH。 该反应的最终结果是增强 母体分子的溶解性、转运和排泄。 的 拟议项目期间的具体目标侧重于三个主要方面， 研究领域包括开发合适的表达系统 对于这两种蛋白质，研究酶的作用机制 使用专门设计的底物，动力学和同位素 标记实验和酶结构的研究 使用物理和生物化学技术。 该委员会的具体目标 关于环氧化物水解酶的项目是：（一）建设和 优化基于细菌或杆状病毒的表达系统， 产生足够数量的蛋白质， 研究;（ii）使用含氮底物类似物 （氮杂芳烃氧化物和氮丙啶）和空间位阻芳烃氧化物， 探讨作用机理及底物去溶剂化作用 识别和催化;（iii）单翻转同位素标记 排除或确认酯参与的实验 催化中间体;（iv）位点特异性诱变以鉴定 （v）流体动力学研究，以确定 天然酶的寡聚状态和N-末端截短形式 作为寻找X射线衍射质量的前奏 EH的单晶。 调查UDP的具体目标- 葡糖醛酸基转移酶包括（i）构建基于杆状病毒的 苯酚UDPGT的表达系统和表征 不成熟和成熟的蛋白质产物和各种截短的蛋白质;（ii） 寻找合适的细菌表达系统;（iii） 选择性底物催化反应的内平衡常数 试图定义"进化最优"底物，和（iv） 探索酶的构象特异性， 二氢二醇底物。 这些研究预计将增加我们的 了解环境代谢的分子细节 污染物和药物，并提高我们预测代谢的能力， 新化合物的情况。

The metabolism and detoxication of potentially toxic foreign (xenobiotic) molecules in eukaryotes relies on the involvement of several membrane- bound enzymes such as cytochrome P-450, epoxide hydrolase and UDP- glucuronosyltransferase that participate in coherent metabolic pathways. To truly comprehend the pathways for the metabolism and detoxication of xenobiotics it is necessary to have a basic understanding of the mechanism of action, substrate specificity and structure of each enzyme that participates or could participate in metabolism. The research program outlined in this proposal seeks to enhance that understanding with respect to two membrane-bound detoxication enzymes, epoxide hydrolase (EH) and UDP-glucuronosyltransferase (UDPGT). Microsomal EH catalyzes the trans-addition of water to epoxides that are often generated by the action of cytochrome P-450 on unsaturated hydrocarbons. UDPGT catalyzes the transfer of the glucuronyl group from UDP-glucuronate to hydrophobic molecules bearing nucleophilic functional groups such as - OH, -SH, -NH2 and -COOH. The net result of this reaction is to enhance the solubility, transport and excretion of the parent molecule. The specific aims for the proposed project period focus on three principal areas of inquiry including the development of suitable expression systems for the two proteins, studies of the mechanism of action of the enzymes using the specifically designed substrates, kinetic, and isotopic labeling experiments, and investigations of the structure of the enzymes using physical and biochemical techniques. The specific aims of the project with respect to epoxide hydrolase are: (i) the construction and optimization of bacterial or baculovirus-based expression systems to produce sufficient quantities of protein for mechanistic and structural studies; (ii) the use of nitrogen-containing substrate analogues (azaarene oxides and aziridines) and sterically hindered arene oxides to probe the mechanism of action and the role of desolvation in substrate recognition and catalysis; (iii) single-turnover isotopic labeling experiments to rule out or confirm the participation f an ester intermediate in catalysis; (iv) site specific mutagenesis to identify putative active site residues; (v) hydrodynamic studies to define the oligomeric state of the native enzyme and N-terminal truncated versions of the enzyme as a prelude to a search for x-ray diffraction-quality single crystals of EH. The specific aims for the investigations of UDP- glucuronosyltransferase include (i) construction of a baculovirus-based expression system for the phenol UDPGT and characterization of the immature and mature protein products and various truncated proteins; (ii) a search for a suitable bacterial expression system; (iii) definition of the internal equilibrium constant in catalysis for selected substrates in an attempt to define an "evolutionarily optimal" substrate and (iv) an exploration of the conformer specificity of the enzyme toward dihydrodiol substrates. These studies are anticipated to increase our understanding of the molecular details of the metabolism of environmental pollutants and drugs and enhance our ability to predict metabolic scenarios for new compounds.