Structural Studies of Thiamin Dependent Enzymes

硫胺素依赖性酶的结构研究

• 批准号: 9112795
• 负责人: Frank Jordan
• 金额: $ 4万
• 依托单位: Rutgers University New Brunswick
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-04-15 至 1993-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9112795&HistoricalAwards=false
• 关键词: Structural; Studies; Thiamin; Dependent; Enzymes

Thiamin diphosphate (TDP, the vitamin B1 coenzyme) is one of the most important coenzymes in carbohydrate metabolism catalyzing e.g. the oxidative decarboxylation of pyruvate to acetylCoA in the reaction catalyzed by the pyruvate dehydrogenase multienzyme complex, enabling the product of glycolysis to enter the citric acid cycle. High resolution structural of such TDP-dependent pyruvate decarboxylating enzymes are being conducted by a variety of techniques. The principal target has been brewers'yeast pyruvate decarboxylase (PDC, E.C. 4 1.1.1) recently purified as fully active alpha4 and beta4 homotetramers in addition to the alpha2beta2 heteroteramer reporter earlier. In a most important and exciting recent development the alpha 4 structure was crystallized in the laboratory already providing novel information, and pointing to the likely success of resolution to at least 2.4 A. PDC is the most widely studied TDP-dependent eznyme and is therefore the best one on which to undertake the detailed structural studies proposed below. In addition to the x-ray developments major progress has been made in the spectroscopic characterization of enzyme-bound intermediates. It is proposed to extend these studies along the following lines of inquiry: 1. structural work on PDC by x-ray crystallographic methods (this part of the research is a collaborative effort with SAX, Fuery and coworkers at the VA Hospital Biocrystallography Lab/Univ. of Pittsburgh)n to determine: a. the structure of the "TDP fold", identify the putative beta-turn-alpha-turn-beta structure at the TDP binding site; b. the basis of interaction between the TDP and Mg2+ with PDC; c./ the conformation of bound TDP, and of putative covalent intermediates; d. the nature of protein-protein interactions in PDC, i.e. among subunits in the absence and presence of the allosteric regulator pyruvamide; 2. mechanistic work of PDC, especially to elucidate the nonoxidative and oxidative chemistry of the enzyme-bound enamine; 3. immunochemical structural studies on PDC to determine immunochemical differences, if any, among isoforms; and on pyruvate dehydrogenase to probe its structure with the monoclones already at hand; 4. structural work on PDC and its TDP fold by NMR methods to determine the microenvironmental factors in catalysis. A collaboration has been initiated with S. Hohmann, who has cloned PDC in a variety of forms, and has and will supply the yeast containing the site-directed mutants, as well as the clones to address both structural and mechanistic questions.

硫胺素二磷酸（Thiamin diphosphate， TDP，维生素B1辅酶）是碳水化合物代谢中最重要的辅酶之一，如丙酮酸脱氢酶多酶复合物催化丙酮酸氧化脱羧生成乙酰辅酶a，使糖酵解产物进入柠檬酸循环。这种tdp依赖性丙酮酸脱羧酶的高分辨率结构正在通过各种技术进行。主要目标是啤酒酵母丙酮酸脱羧酶（PDC， E.C. 4 1.1.1）最近被纯化为完全活性的α 4和β 4同四聚体，以及早期的α 2 β 2异四聚体报告物。在最近一项最重要和令人兴奋的发展中，α 4结构在实验室中结晶化，已经提供了新的信息，并指出可能成功地解决至少2.4 a的问题。PDC是研究最广泛的tdp依赖性eznyme，因此是进行下面提出的详细结构研究的最佳选择。除了x射线的发展外，酶结合中间体的光谱表征也取得了重大进展。建议沿着以下调查路线扩展这些研究：通过x射线晶体学方法对PDC进行结构研究(这部分研究是与弗吉尼亚州医院生物晶体学实验室/大学的SAX， Fuery和同事合作完成的。a.确定“TDP折叠”的结构，确定TDP结合位点推定的β -turn- α -turn- β结构；b. TDP、Mg2+与PDC相互作用的基础；c./结合TDP的构象，以及假定的共价中间体；d. PDC中蛋白质-蛋白质相互作用的性质，即在没有变构调节剂丙酮酰胺的情况下亚基之间的相互作用；2. PDC的机制工作，特别是阐明酶结合的烯胺的非氧化和氧化化学；3. 免疫化学结构研究PDC以确定免疫化学差异（如果有的话）；以及丙酮酸脱氢酶，用已有的单克隆体探测其结构；4. 利用核磁共振方法对PDC及其TDP折叠进行结构研究，确定催化过程中的微环境因素。已经开始与S. Hohmann合作，他已经克隆了多种形式的PDC，并且已经并将提供含有位点定向突变体的酵母，以及解决结构和机制问题的克隆。