STRAINED INTERMEDIATES IN ENZYME CATALYZED REACTIONS

X射线晶体学提供了衬底的原子分辨率视图 和结合在酶活性位点的抑制剂，传达了这样的印象 活性位点官能团提供了精确对齐的 配体的“溶剂化球”。 然而，这些结构的分辨率 不足以确定这种特定溶剂化 改变了结合分子的化学反应性。 中的实验 该建议旨在检测和量化电子应变， 存在于与烯酰辅酶A水合酶、乳酸脱氢酶 和3-羟基-3-甲基戊二酰辅酶A还原酶。 主要方法是 使用的是光谱学，主要是共振拉曼和UV，以及同位素 方面的影响. 这些研究将增加我们对酶的理解 为过渡态的设计提供必要的背景 类似物 已提出巴豆酸酶催化协同的顺式消除 反应 新的H/D/T同位素效应实验将证实 协调一致的机制，并确定过渡国家是否是 其特征在于隧道效应。 我们将进一步研究 底物和产物复合物的基态结构 光谱学上 初步的紫外和共振拉曼结果表明 束缚态的电子结构会发生剧烈的变化 印刷受体. 催化顺式异构体消除的酶是一类不断增长的 酶，包括一些核酸内切酶，没有很好地表征 机械地。 过去的实验研究忽略了羧酰胺在 脱氢酶反应 乳酸盐的极端立体专一性 脱氢酶将用位点特异性突变体测定， 核苷酸类似物，以量化羧酰胺在 催化机理 二氢吡啶环中应变的诱导 将通过测量同位素来检查三元复合物中的NADH 对协会的影响。 定义明确的离子对和氢键 酶与乳酸和草氨酸的相互作用将使我们能够 校准这些分子相互作用对同位素效应的影响 关于协会。 HMG-CoA还原酶是一种药学上重要的酶， 最近被克隆，过度表达和结晶。 的二硫酯 HMG-CoA是该酶的有效抑制剂，其另外诱导 NAD（P）H底物抑制。 我们计划研究立体化学， 这种酶的光谱和动力学研究，以描绘 硫代羰基极化对异常抑制的重要性。

X-ray crystallography has provided atomic resolution views of substrates and inhibitors bound at enzyme active sites, conveying the impression that the active site functional groups provide a precisely aligned "solvation sphere" for the ligand. Yet, the resolution of the structures is insufficient to determine the extent that this specific solvation alters the chemical reactivity of the bound molecule. The experiments in this proposal are designed to detect and quantify the electronic strain present in substrates bound to enoyl-CoA hydratase, lactate dehydrogenase and 3-hydroxy-3-methylglutaryl-CoA reductase. The primary methods to be used are spectroscopic, largely resonance Raman and UV, and isotope effects. The proposed studies will increase our understanding of enzyme catalysis and provide the necessary background to design transition state analogs. Crotonase has been proposed to catalyze a concerted syn elimination reaction. Novel H/D/T isotope effect experiments that will confirm the concerted mechanism and establish whether the transition state is characterized by tunneling are proposed. Further we will examine the ground state structure of the substrate and product complexes spectroscopically. Preliminary UV and resonance Raman results indicate there are dramatic alterations in the electronic structure of the bound substrates. Enzymes catalyzing syn eliminations are a growing class of enzyme, including some endonucleases, that are not well characterized mechanistically. Past experimental studies have ignored the function of the carboxamide in dehydrogenase reactions. The extreme stereospecificity of lactate dehydrogenase will be determined with site specific mutants and nucleotide analogs to quantify the importance of the carboxamide in the catalytic mechanism. The induction of strain in the dihydropyridine ring of NADH in the ternary complex will be examined by measuring isotope effects on association. The well defined ion pair and H-bonding interactions of the enzyme with lactate and oxamate will allow us to calibrate the effects of these molecular interactions on isotope effects on association. HMG-CoA reductase is a pharmaceutically important enzyme that has recently been cloned, overexpressed and crystallized. The dithioester of HMG-CoA is a potent inhibitor of this enzyme, which additionally induces substrate inhibition by NAD(P)H. We plan to pursue stereochemical, spectroscopic and kinetic studies of this enzyme to delineate the importance of the thiocarbonyl polarization to the unusual inhibition.