MEMBRANE-BOUND DETOXICATION ENZYMES

潜在毒性外来(异物)的代谢与解毒 真核生物中的分子依赖于几个膜的参与- 结合酶如细胞色素P-450、环氧化物水解酶和UDP- 参与连贯代谢途径的葡萄糖醛酸基转移酶。 要真正了解人体新陈代谢和解毒的途径 有必要对外来生物有一个基本的了解 各酶的作用机制、底物专一性和结构 参与或可能参与新陈代谢的人。这项研究 本提案中概述的计划旨在加强这一理解 关于两种膜结合解毒酶，环氧化物 水解酶(EH)和UDP-葡萄糖醛酸基转移酶(UDPGT)。微粒体型EH 催化水与环氧化物的反式加成反应 由细胞色素P-450作用于不饱和碳氢化合物而产生。 UDPGT催化UDP-葡萄糖醛酸基转移 到含有亲核官能团的疏水分子，例如- 哦，-SH，-NH2和-COOH。这种反应的最终结果是增强 母体分子的溶解、运输和排泄。这个 拟议项目期的具体目标集中在三个主要方面 研究领域，包括开发合适的表达系统 对于这两种蛋白质，酶的作用机理进行了研究 使用专门设计的底物、动力学和同位素 标记实验和酶的结构研究 使用物理和生化技术。《公约》的具体目标 与环氧化物水解酶有关的项目是：(I)建造和 细菌或杆状病毒表达系统的优化 为机械和结构产生足够数量的蛋白质 研究；(2)使用含氮底物类似物 (氮杂芳烃氧化物和氮杂环丙烷)和空间受阻的芳烃氧化物 探讨脱溶在底物中的作用机理和作用 识别和催化；(3)单周转同位素标记 排除或确认酯的参与的实验 催化中的中间体；(Iv)定点突变以鉴定 假定的活性中心残留物；(V)流体动力学研究，以确定 天然酶和N-末端截短型的低聚状态 作为寻找x射线衍射质量的前奏 EH的单晶。UDP调查的具体目标是- 葡萄糖醛酸基转移酶包括(I)杆状病毒载体的构建 苯酚UDPGT的表达系统及其特性研究 未成熟和成熟的蛋白质产品和各种截短的蛋白质； 寻找合适的细菌表达系统；(Iii)定义 选定底物在催化中的内平衡常数 试图定义一种“进化上最优”的底物和(Iv) 对酶的构象特异性的探讨 二氢二醇底物。这些研究预计将增加我们的 了解环境新陈代谢的分子细节 污染物和药物，提高我们预测新陈代谢的能力 新化合物的情景。

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.