SPECTROSCOPIC STUDIES OF ENZYME/SUBSTRATE COMPLEXES

酶/底物复合物的光谱研究

• 批准号: 2178055
• 负责人: GEORGE H REED
• 金额: $ 20.71万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-01-01 至 1998-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2178055
• 关键词: X ray crystallography; active sites; adenosine diphosphate; adenosine triphosphate; carbanion; catalyst; cations; cofactor; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate analog; enzyme substrate complex; glycolysis; magnesium; mass spectrometry; mutant; nitro compounds; nuclear magnetic resonance spectroscopy; phosphoenolpyruvate; phosphopyruvate hydratase; pyruvate kinase; stereochemistry; yeasts

The objectives of this proposal are to perform structural and mechanistic analyses on two enzymes from glycolysis, rabbit muscle pyruvate kinase and yeast enolase. Both of these enzymes require multiple equivalents of inorganic cations for their function, and the means by which the metal ions promote activity are not fully understood. Crystal structures which were solved for both of these enzymes during the past budget period, together with structural information obtained from EPR spectroscopic studies, provide the background for the proposed experiments. Clones of genes for both enzymes are available, and site specific mutagenesis can be used to augment the structural studies. For pyruvate kinase, we have crystals for X-ray structural analysis of the fully liganded form of the enzyme-including a form with authentic ATP bound in the active site. The present X-ray structure suggests a plausible mechanism for the monovalent cation activation of the enzyme, and this mechanism will be tested by site specific mutants of residues in the vicinity of the monovalent cation site. Candidates for the general acid/base catalyst, revealed in the structure, will be screened by mutations and structure/function analyses of the mutant enzymes. For enolase, our structure for a new, high pH, low salt, crystal form of the enzyme with two Mg ions and an intermediate state analog bound in the active site indicates that there are stereochemical inconsistencies in the current model of enolase catalysis. We will approach this issue by obtaining crystal structures of bis metal ion complexes of the -enzyme-substrate mixture and of a nitro analog of the proposed carbanion intermediate in the reaction. These studies will be augmented by mutations of specific residues believed to be essential in the two alternative mechanisms and by systematic analyses of the effects of these mutations. The functional importance of the closure of an intricate active site flap will be examined by compromising flap closure through mutagenesis.

该提案的目标是执行结构和机械 糖酵解中两种酶的分析，兔肌肉丙酮酸激酶 和酵母烯醇化酶。这两种酶都需要多当量 无机阳离子的功能，以及金属的作用方式 离子促进活性尚不完全清楚。晶体结构其中 在过去的预算期内这两种酶都得到了解决， 连同从 EPR 光谱获得的结构信息 研究，为拟议的实验提供背景。的克隆 两种酶的基因都是可用的，并且可以进行位点特异性诱变 用于增强结构研究。 对于丙酮酸激酶，我们有 用于全配位形式的 X 射线结构分析的晶体 酶——包括在活性位点结合有真正的 ATP 的形式。这 目前的 X 射线结构表明单价的可能机制 酶的阳离子激活，该机制将通过 单价附近残基的位点特异性突变体 阳离子位点。一般酸/碱催化剂的候选者，揭示于 结构，将通过突变和结构/功能进行筛选 突变酶的分析。对于烯醇化酶，我们的结构是一种新的、 高 pH、低盐、具有两个 Mg 离子和一个 活性位点中结合的中间态类似物表明存在 当前烯醇酶模型中存在立体化学不一致吗 催化。我们将通过获得晶体结构来解决这个问题 酶-底物混合物的双金属离子络合物和 反应中提议的碳负离子中间体的硝基类似物。 这些研究将通过特定残基的突变而得到加强 被认为在两种替代机制中至关重要 系统分析这些突变的影响。功能性的 关闭复杂的活性部位皮瓣的重要性将是 通过诱变损害皮瓣闭合来检查。