Spectroscopic Insights into the Mechanism of Enoyl-CoA Hydratase

9604254第一部分-技术Enoyl-CoA水合酶催化水在(，(-)不饱和脂肪酸硫醇酯的C-C键上加成。共轭底物类似物与酶的结合伴随着共轭分子的电子结构的实质性变化。PI描述了一系列实验，以研究所观察到的底物类似物的电子结构变化与酶催化(，(-)不饱和乙烯键水合的能力之间的直接关系。最近描述的4-氯苯甲酰辅酶A脱卤酶的X射线晶体结构，是一种与水合酶28%相同，50%相似的酶，为剖析该酶的催化机理提供了一个独特的机会。为了研究底物极化与酶催化的直接关系，将通过定点突变对底物周围的残基进行系统的修饰。然后将对每个突变蛋白质进行组合光谱-反应性研究，以评估突变对催化和底物极化的影响。这些实验将有助于建立酶-底物复合体的直接结构-反应相关性，并有望提供对静电学在酶催化中的作用的总体洞察。第2部分-非技术酶是起生物催化剂作用的蛋白质，可将化学反应速度加快许多数量级。人们对了解酶的工作原理和确定酶活性的基本基础有浓厚的兴趣。这项提议的重点是Enoyl-CoA水合酶，这是一种参与体内脂肪酸分解的酶。利用定点突变，PI计划识别和分析负责反应化学步骤的酶活性部位的氨基酸残基。这一建议的一个具体目标是将活性中心中底物的结构与反应速度定量地结合起来。结构信息将使用拉曼光谱和核磁共振光谱提供。此外，他还打算通过突变和监测对底物结构和反应性的影响来阐明蛋白质‘电场’的作用。这些信息不仅对理解Enoyl-CoA水合酶的机制有重要价值，而且还将提供对静电力在酶催化中的作用的直接洞察。

9604254 Tonge Part 1-Technical The enzyme enoyl-CoA hydratase catalyzes the addition of water across the C-C bond of (,(-unsaturated fatty acid thiol esters. Binding of conjugated subtrate analogs to the enzyme is accompanied by substantial alterations in the electronic structure of the conjugated molecules. The PI describes a series of experiments to investigate the direct relationship between the observed alteration in electronic structure of the substrate analogs and the enzyme's ability to catalyze the hydration of (,(-unsaturated ethylenic bond. The recently described X-ray crystal structure of 4-chlorobenzoyl-CoA dehalogenase, an enzyme that is 28% identical and 50% similar to the hydratase, presents a unique opportunity to dissect the catalytic mechanism of the enzyme. In order to investigate the direct relationship between substrate polarization and enzyme catalysis, residues around the substrate will be systematically modified by site-directed mutagenesis. Combined spectroscopic-reactivity studies will then be performed on each mutant protein to assess the effects of the mutations on catalysis and substrate polarization. These experiments will facilitate the establishment of a direct structure-reactivity correlation for the enzyme-substrate complex and are expected to provide general insight into the role of electrostatics in enzyme catalysis. Part 2- Non Technical Enzymes are proteins that function as biological catalysts, accelerating the rates of chemical reactions by many orders of magnitude. There is intense interest in understanding how enzymes work and in identifying the fundamental basis for enzyme activity. The focus of this proposal is enoyl-CoA hydratase, an enzyme involved in the breakdown of fatty acids in the body. Using site directed mutagenesis, the PI plans to identify and analyze the amino acid residues in the active site of the enzyme that are responsible for the chemical steps of the reaction. A specific goal of this proposal is to quantitatively re late the structure of the substrate in the active site to the rate of the reaction. The structural information will be provided using Raman and NMR spectroscopy. In addition he intends to elucidate the role of the protein's 'electric field' through mutagenesis and monitoring the effect on substrate structure and reactivity. Not only will this information be of great value in understanding the mechanism of enoyl-CoA hydratase, but it will also provide direct insight into the role of electrostatic forces in enzyme catalysis.