MECHANISMS OF ENZYMIC DETOXICATION REACTIONS

本研究的目的在于阐明 谷胱甘肽S-转移酶和尿苷-5的作用 在化学和物理上 有毒外来化合物（异生物质）的处置。 亲电 底物（芳烃氧化物、β-内酯和α，β-不饱和脂肪酸） 化合物）和亲核底物（芳香族化合物的邻位二氢二醇）。 已知区域和立体化学的烃）将用于绘制 相对非特异性亲脂性结合位点的拓扑结构 谷胱甘肽S-转移酶和UDP-葡萄糖醛酸转移酶。 作为谷胱甘肽详细稳态动力学研究的前奏 S-转移酶，几种谷胱甘肽类似物，包括γ-L-Glu-L-SerGly， γ-L-Glu-L-AlaGly，还原型谷胱甘肽，和两种亚甲基同系物， β-L-CysGly和γ-L-Glu-L-Cys-Beta-Ala将在 量用作替代底物和死端抑制剂。 催化机理的化学研究将包括尝试 沿反应坐标沿着捕获过渡态或中间体， 谷胱甘肽与环氧化物和芳烃氧化物的反应。 电离 结合谷胱甘肽的行为将通过pH依赖性的研究， 稳态动力学和酶结合pKa的直接测定 将通过13 C-NMR光谱法尝试谷胱甘肽。 的能力 谷胱甘肽S-转移酶转运亲脂性溶质之间 将研究脂质体群体以评估其可能的 作为细胞内异生物质转运蛋白的作用。 化学和 UDP-葡萄糖醛酸基转移酶动力学机制将通过 使用尿苷-5的α-仲氘动力学同位素效应 在异头碳上氘代的足二磷酸葡萄糖醛酸， 稳态动力学研究采用非反应性亚甲基类似物的 UDP-葡萄糖醛酸。 动力学研究将用于探测磷脂 依赖性和二价金属离子依赖性的酶。 研究 旨在深入了解成功的分子细节， 谷胱甘肽S-转移酶和UDP-葡萄糖醛酸基转移酶在 异生物质的代谢。

The research is directed at the elucidation of the role and mechanisms of action of glutathione S-transferase and uridine-5 feet-diphosphoglucuronosyltransferase in the chemical and physical disposition of toxic foreign compounds (xenobiotics). Electrophilic substrates (arene oxides, Beta-lactones and Alpha, Beta-unsaturated compounds) and nucleophilic substrates (vicinal dihydrodiols of aromatic hydrocarbons) of known regio- and stereochemistry will be used to map the topology of the relatively nonspecific lipophilic binding sites of glutathione S-transferase and UDP-glucuronosyltransferase, respectively. As a prelude to detailed steady-state kinetic studies of glutathione S-transferase, several glutathione analogs, including Gamma-L-Glu-L-SerGly, Gamma-L-Glu-L-AlaGly, retroglutathione, and two methylene homologs, Beta-L-CysGly and Gamma-L-Glu-L-Cys-Beta-Ala, will be synthesized in quantity for use as alternative substrates and dead-end inhibitors. Chemical studies of the mechanism of catalysis will include attempts to trap transition states or intermediates along the reaction coordinate, in the reaction of glutathione with epoxides and arene oxides. The ionization behavior of bound glutathione will be studied by the pH dependence of the steady-state kinetics and a direct determination of the pKa of enzyme bound glutathione will be attempted by 13C-NMR spectroscopy. The ability of glutathione S-transferase to transport lipophilic solutes between populations of liposomes will be studied in order to assess its possible role as an intracellular xenobiotic transport protein. The chemical and kinetic mechanism of UDP-glucuronosyltransferase will be probed by Alpha-secondary deuterium kinetic isotope effects using uridine-5 feet-diphosphoglucuronate deuterated at the anomeric carbon and by steady-state kinetic studies employing a non-reactive methylene analog of UDP-glucuronate. Kinetic studies will be used to probe the phospholipid dependence and divalent metal ion dependence of the enzyme. The studies are designed to gain insight into the molecular details of the success or failure of glutathione S-transferase and UDP-glucuronosyltransferase in the metabolism of xenobiotics.