Spectroscopic Insights into the Mechanism of Enoyl-CoA Hydratase

烯酰辅酶 A 水合酶机制的光谱洞察

• 批准号: 9604254
• 负责人: Peter Tonge
• 金额: $ 29.8万
• 依托单位: SUNY at Stony Brook
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-03-15 至 2001-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9604254&HistoricalAwards=false
• 关键词: Spectroscopic; Insights; into; Mechanism; Enoyl

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.

9604254通格第1部分-技术该酶烯酰辅酶A水合酶催化穿过（，）-不饱和脂肪酸硫酯的C-C键的水加成。 结合的底物类似物的酶是伴随着在共轭分子的电子结构的实质性改变。 PI描述了一系列实验，以研究观察到的底物类似物的电子结构变化与酶催化（，（-不饱和烯键的水合作用的能力之间的直接关系。 最近描述的4-氯苯甲酰基辅酶A脱卤酶的X-射线晶体结构，是一种酶，是28%相同和50%相似的水合酶，提供了一个独特的机会来剖析酶的催化机制。 为了研究底物极化与酶催化之间的直接关系，将通过定点突变系统地修饰底物周围的残基。 然后将对每种突变蛋白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一个直接的结构-反应性相关的酶-底物复合物，并预计提供一般的洞察力在酶催化中的作用的静电。 第2部分-非技术酶是作为生物催化剂的蛋白质，将化学反应的速率加速许多数量级。 人们对了解酶如何工作和确定酶活性的基本基础有着浓厚的兴趣。 该提案的重点是烯酰辅酶A水合酶，一种参与体内脂肪酸分解的酶。 使用定点诱变，PI计划鉴定和分析酶活性位点中负责反应化学步骤的氨基酸残基。 该提议的一个具体目标是定量地将活性位点中底物的结构与反应速率联系起来。 将使用拉曼和NMR光谱提供结构信息。 此外，他打算通过诱变和监测对底物结构和反应性的影响来阐明蛋白质的“电场”的作用。 这些信息不仅对了解烯酰辅酶A水合酶的作用机制具有重要价值，而且还将直接深入了解静电力在酶催化中的作用。