ISOTOPIC PROBES OF ENZYMATIC REACTION MECHANISMS

本文所述研究的广泛、长期目标 这些建议旨在阐明复杂的关系 结构和功能之间的联系 主要重点 本申请的第一部分涉及催化机理， 细菌磷酸三酯酶的结构。 这种酶催化 解毒或有机磷神经毒素通过水解 磷-氧键和磷-氟键。 磷酸三酯酶 在扩散时催化最佳底物的水解， 控制限度和二价阳离子需要催化 活动 基本金属离子的作用将通过以下方法来探讨： 用各种光谱学方法取代天然Zn 2+离子， 活性阳离子（Cd 2+、Mn 2+、Ni 2+、Co 2+和Cu 2+）。 配体 金属位点的环境将通过113 Cd-NMR来解决 谱 金属位点和基质之间的相互作用将 使用顺磁性金属离子的ESR光谱获得 衍生物. 光谱研究将得到补充， 潜在的氨基酸配体（组氨酸，半胱氨酸， 天冬氨酸和谷氨酸）。 的 突变蛋白将用于序列特异性分配 蛋白质配体的金属中心，并作为探针的作用， 这些金属离子在催化事件中起作用。 身份和功能 将测定该酶活性部位的其它氨基酸的量 通过炔基磷酸酯的设计和合成。 这些 硅化物底物与活性位点亲核试剂共价反应， 导致催化作用完全失活。 重原子氧-18 使用野生型和突变体的快速和慢速底物的同位素效应 蛋白质将被用来确定过渡的分布 国家结构。 X射线晶体学分析将开始， 努力确定折叠的三维结构 蛋白

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.