STRAINED INTERMEDIATES IN ENZYME CATALYZED REACTIONS

X射线结晶学已经提供了衬底的原子分辨率视图。 以及结合在酶活性部位的抑制剂，传达这样的印象 活动站点功能组提供了精确一致的 配位体的“溶剂化球体”。然而，这些结构的分辨率 不足以确定这种特定的溶剂化作用的程度 改变结合分子的化学反应能力。在中国的实验 这项建议旨在检测和量化电子应变 存在于与Enoyl-CoA水合酶、乳酸脱氢酶结合的底物中 3-羟基-3-甲基戊二酰辅酶A还原酶。主要的方法是 使用的是光谱，主要是共振拉曼和紫外，以及同位素 效果。建议的研究将增加我们对酶的了解。 催化作用，并为设计过渡态提供必要的背景 类比。 巴豆酶已被提出用来催化一种协同的联氨消除。 反应。新的H/D/T同位素效应实验将证实 协调机制，确定过渡状态是否为 提出了以隧道效应为特征的光传输协议。此外，我们还将研究 底物和产物络合物的基态结构 从光谱上看。初步的UV和共振拉曼结果表明 束缚的电子结构发生了戏剧性的变化 底物。催化SYN消除的酶是一类不断增长的 酶，包括一些没有很好描述的核酸内切酶 从机械上讲。 以往的实验研究忽略了羧胺在体内的作用。 脱氢酶反应。乳酸的极端立体专一性 脱氢酶将通过特定位点的突变体和 用核苷酸类似物来量化羧胺在 催化机理。二氢吡啶环上菌株的诱导 将通过测量同位素来检测三元络合物中的NADH 对联想的影响。定义明确的离子对与氢键 这种酶与乳酸和草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 在协会上。 HMG-CoA还原酶是一种重要的药用酶，它具有 最近被克隆、过度表达和结晶。的二硫代酯 HMG-CoA是这种酶的有效抑制剂，它还可以诱导 NAD(P)H对底物的抑制我们计划追求立体化学， 该酶的光谱和动力学研究，以描绘 硫代羰基极化对不寻常抑制的重要性。

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.