MECHANISMS OF ENZYMIC DETOXICATION REACTIONS

酶解毒反应的机制

• 批准号: 3072681
• 负责人: RICHARD N ARMSTRONG
• 金额: $ 5.25万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 1989-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3072681
• 关键词: X ray spectrometry; binding proteins; conformation; detoxification; deuterium; enzyme inhibitors; enzyme mechanism; enzyme substrate analog; epoxides; glucuronosyltransferase; glutathione transferase; high performance liquid chromatography; isozymes; laboratory rat; molecular site; nuclear magnetic resonance spectroscopy; oxides; peptide chemical synthesis; phospholipids; radionuclide double label; radionuclides; spectrometry; stereochemistry; toxicant interaction; transport proteins; tritium

The research is directed at the elucidation of the role and mechanisms of action of glutathione S-transferase and uridine-5'- prime-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'- 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.

这项研究旨在阐明其作用和机制。 谷胱甘肽S转移酶和尿苷-5‘-的作用 初级-二磷酸葡萄糖醛酸基转移酶在化学和物理中的作用 有毒外来化合物(外来生物物质)的处置。亲电的 底物(芳烃氧化物、β-内酯和α、β-不饱和 化合物)和亲核底物(芳香族邻位二氢二醇 已知区域和立体化学的碳氢化合物)将被用来绘制 的相对非特异性亲脂结合部位的拓扑结构 谷胱甘肽S转移酶和UDP葡萄糖醛酸基转移酶。 作为详细的谷胱甘肽稳态动力学研究的前奏 S-转移酶，几种谷胱甘肽类似物，包括伽马-L-谷氨酸-L-Sergly， 伽马-L-谷氨酸-L-丙氨酸、还原型谷胱甘肽和两个亚甲基同系物， β-L-半胱氨酸和伽马-L-谷氨酸-L-半胱氨酸-β-丙氨酸，将在 用作替代底物和末端抑制剂的数量。 对催化机理的化学研究将包括尝试 沿反应坐标的陷阱过渡态或中间体，In 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依赖关系来研究 酶结合的稳态动力学及pKa的直接测定 谷胱甘肽将通过~(13)C-核磁共振波谱进行尝试。的能力 谷胱甘肽S转移酶在亲脂性溶质间的转运 将对脂质体的种群进行研究，以评估其可能性 作为细胞内异种运输蛋白的作用。化学物质和 UDP-葡萄糖醛酸基转移酶的动力学机制 尿苷-5‘-的α-二次氚动力学同位素效应 二磷酸葡萄糖尿酸在非均相碳上和通过 采用非反应性亚甲基类似物的稳态动力学研究 UDP-葡萄糖醛酸脂。动力学研究将用于探测磷脂 该酶的依赖性和二价金属离子依赖性。这些研究 旨在洞察成功或失败的分子细节 谷胱甘肽S转移酶和尿苷二磷酸葡萄糖醛酸基转移酶失活 外源生物的代谢。