ISOTOPIC PROBES OF ENZYMATIC REACTION MECHANISMS

酶反应机制的同位素探针

• 批准号: 2177177
• 负责人: Frank M. Raushel
• 金额: $ 17.13万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-12-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2177177
• 关键词: X ray crystallography; active sites; bacterial proteins; cadmium; chemical binding; chemical kinetics; chemical substitution; cobalt; copper; crystallization; divalent cations; electron spin resonance spectroscopy; enzyme complex; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate analog; esterase; esterase inhibitor; histidine; hydrolysis; ligands; manganese; metalloenzyme; mutant; nuclear magnetic resonance spectroscopy; oxygen; protein sequence; site directed mutagenesis; stable isotope; zinc

The broad, long-term objectives for the research described in this proposal are aimed at the elucidation of the complex relationships between structure and function in biological systems. The primary focus of the present application is directed at the mechanism of catalysis and structure of the bacterial phosphotriesterase. This enzyme catalyzes the detoxification or organophosphate neurotoxins through the hydrolysis of phosphorus-oxygen and phosphorus-fluorine bonds. The phosphotriesterase catalyzes the hydrolysis of optimal substrates at the diffusion- controlled limit and a divalent cation is required for catalytic activity. The role of the essential metal ion will be probed by substitution of the native Zn2+ ion with a variety of spectroscopically active cations (Cd2+, Mn2+, Ni2+, Co2+, and Cu2+). The ligand environment of the metal sites will be addressed by 113Cd-NMR spectroscopy. Interactions between the metal sites and substrates will be obtained using ESR spectroscopy with the paramagnetic metal ion derivatives. The spectroscopic studies will be complemented by replacement of potential amino acid ligands (histidine, cysteine, aspartate, and glutamate) using site-directed mutagenesis protocols. The mutant proteins will be utilized for the sequence specific assignments of the protein ligands to the metal centers and as probes for the role these metal ions play in the catalytic events. The identity and function of other amino acids at the active site of this enzyme will be determined through the design and synthesis of alkynyl phosphate esters. These suicide substrates covalently react with active site nucleophiles and result in complete inactivation of catalysis. Heavy atom oxygen-18 isotope effects with fast and slow substrates using wild type and mutant proteins will be utilized to determine the distribution of transition state structures. An X-ray crystallographic analysis will begin in an effort to determine the three-dimensional structure of the folded protein.

在此描述的研究的广泛，长期目标 提案旨在阐明复杂关系 在生物系统中的结构和功能之间。 主要重点 本应用的目的是针对催化机制和 细菌磷酸酯酶的结构。 这种酶催化了 解毒或有机磷酸神经毒素通过水解的水解 磷 - 氧和磷氟键。 磷酸三酯酶 催化在扩散 - 最佳底物的水解 - 催化需要受控的极限和二价阳离子 活动。 必需金属离子的作用将由 用各种光谱替代天然Zn2+离子 活性阳离子（CD2+，MN2+，Ni2+，Co2+和Cu2+）。 配体 金属站点的环境将由113CD-NMR解决 光谱法。 金属位点和基材之间的相互作用将 使用副磁性金属离子使用ESR光谱获得 衍生物。 光谱研究将由 取代潜在氨基酸配体（组氨酸，半胱氨酸， 天冬氨酸和谷氨酸）使用定向诱变方案。 这 突变蛋白将用于序列特定分配 蛋白质配体的金属中心，并作为该角色的探针 这些金属离子在催化事件中发挥作用。 身份和功能 将确定该酶的活性位点的其他氨基酸 通过烷基磷酸酯的设计和合成。 这些 自杀底物与活性部位亲核试剂共价反应 导致催化完全失活。 重原子氧18 使用野生型和突变体的快速和缓慢基板的同位素效应 蛋白质将用于确定过渡的分布 国家结构。 X射线晶体学分析将以 努力确定折叠的三维结构 蛋白质。