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 分析糖酵解，兔肌肉丙酮酸激酶的两种酶 和酵母烯醇酶。这两种酶都需要多个等效的 无机阳离子的功能以及金属的手段 离子促进活动尚未完全理解。晶体结构 在过去的预算期间，这两种酶都被解决了， 以及从EPR光谱获得的结构信息 研究，为提出的实验提供了背景。克隆 两种酶的基因都可以使用，特定位点的诱变可以 用于增加结构研究。 对于丙酮酸激酶，我们有 晶体进行X射线结构分析的完全配体形式的晶体 酶在活性位点中绑定了具有正宗ATP的形式。这 当前的X射线结构提出了单价的合理机制 酶的阳离子激活，该机制将通过 单价附近残留物的位点特异性突变体 阳离子网站。一般酸/碱催化剂的候选物，在 该结构将通过突变和结构/功能筛选 突变酶的分析。对于烯醇酶，我们的新结构 高pH，低盐，晶体的晶体形式，带有两个mg离子和一个 活动站点中绑定的中间状态模拟表明 在当前的烯醇酶模型中是立体化学不一致之处 催化。我们将通过获得晶体结构来解决这个问题 - 酶 - 基底混合物和A 反应中提出的carbanion的硝基类似物。 这些研究将通过特定残基的突变增强 相信在两种替代机制中至关重要 这些突变影响的系统分析。功能 复杂的活动场地襟翼关闭的重要性将是 通过通过诱变来损害皮瓣闭合来检查。