Structure and Mechanism of alpha-Ketoacid Decarboxylases and Dehydrogenases

α-酮酸脱羧酶和脱氢酶的结构和机制

• 批准号: 7783217
• 负责人: FRANK JORDAN
• 金额: $ 37.03万
• 依托单位: RUTGERS THE STATE UNIV OF NJ NEWARK
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7783217
• 关键词: 4-aminopyrimidine; Abbreviations; Acetaldehyde; Acetyl Coenzyme A; Acids; Address; Azotobacter vinelandii; Bacillus stearothermophilus; Bacteria; Behavior; Binding; Carbon Dioxide; Carboxy-Lyases; Catalysis; Cells; Chemicals; Citric Acid Cycle; CoASH; Cocos; Coenzymes; Collaborations; Complex; Coupling; Crystallography; Cysteine; Detection; Diabetes Mellitus; Electronics; Enzymatic Biochemistry; Enzymes; Escherichia coli; Event; Family; Fluorescence; Glycolysis; Goals; Gram-Positive Bacteria; Grant; Hand; Human; Individual; Jordan; Keto Acids; Kinetics; Label; Laboratories; Malignant Neoplasms; Measures; Metabolism; Methodology; Methods; Monitor; Motion; Movement; Multienzyme Complexes; Mycobacterium tuberculosis; Nature; Oxidoreductase; Pathway interactions; Phase; Phosphoric Monoester Hydrolases; Phosphotransferases; Positioning Attribute; Property; Proteins; Protons; Publications; Publishing; Pyruvate; Pyruvate Decarboxylase; Pyruvate Dehydrogenase Complex; Pyruvates; Pyruvic Acid; Reaction; Recruitment Activity; Regulation; Research; Roentgen Rays; Role; Saccharomyces cerevisiae; Scheme; Site; Solutions; Specificity; Structure; System; Technology; Testing; Thermodynamics; Thiamine; Thiamine Pyrophosphate; Time; Variant; Visit; Work; Yeasts; carbanion; career; innovation; insight; interest; ionization; member; programs; public health relevance; pyruvate dehydrogenase; research study; solid state nuclear magnetic resonance; tautomer; time use; tool; ylide

DESCRIPTION (provided by applicant): This is a request for renewal of a research program on thiamin diphosphate-dependent enzymes, principally on the E. coli and human pyruvate dehydrogenase multienzyme complexes. These complexes are at a key junction in metabolism of virtually all cells, converting the product of glycolysis pyruvic acid to acetyl coenzyme A at the entry to the Krebs cycle (also known as the tricarboxylic acid or citric acid cycle). Goals for the next phase of the project are: (1) Determination of rate-limiting steps, states of ionization and tautomerization of enzyme-bound thiamin-related intermediates, with the intent to compare these properties in isolated components of the complexes to those in the 4,600,000 and 10,000,000 Da complexes. In innovation requiring de novo total synthesis of specifically labeled thiamins, the PI proposes to use solid state NMR, as well as advanced solution NMR methods to accomplish the goals. (2) Exploration of the hypothesis that the mobility of active center loops in thiamin enzymes is correlated with catalysis. With methods published by the PI in 2008, key loops on the E. coli complex's E1 and E2 components have been identified for these studies, including the lipoyl domain of the E2 component, which 'visits' the active centers of all three components. (3) Examination of the structural and functional consequences of assembly in the bacterial and human pyruvate dehydrogenase complexes. In the E. coli complex, the issue to be resolved is whether the same region(s) of the E2 component recognizes the E1 and E3 components, to address the hypothesis that the loci of recognition of E2 for E1 or E3 are different. In the human complex, we wish to determine the regions of E2 component interacting with the E1 component, never accomplished before The methods used by the PI could answer questions that no other current methodology can, such as the definition of proton positions on the 4'-aminopyrimidine ring of the coenzyme at distinct intermediates along the reaction coordinate, a key issue in understanding proton transfers. A group of outstanding collaborators have been recruited for experimental expertise not available in the PI's laboratory, among them M. Patel, a CoPI selected for expertise on the human enzyme, and W. Furey, a long-term collaborator in a string of structure determinations. Results from two complexes will enable the PI to draw general conclusions regarding the entire superfamily of such enzymes, as already demonstrated in several of his recent publications. PUBLIC HEALTH RELEVANCE: This is a request for renewal of a research program on thiamin diphosphate dependent enzymes, principally on the E. coli and human pyruvate dehydrogenase multienzyme complexes. These complexes are at a key junction in metabolism of virtually all cells, converting the product of glycolysis pyruvic acid to acetyl coenzyme A at the entry of the Krebs cycle (also known as the tricarboxylic acid or citric acid cycle). Goals for the next phase of the project include: (1) Determination of rate-limiting steps, states of ionization and tautomerization of enzyme-bound thiamin-related intermediates, with the intent to compare these properties in isolated components of the complexes to those in the 4,600,000 and 10,000,000 Da complexes. (2) Explore the hypothesis that the mobility of active center loops in thiamin enzymes is correlated with catalysis. (3) Examine the structural and functional consequences of assembly in the bacterial and human pyruvate dehydrogenase complexes. In the E. coli complex, the issue to be resolved is whether the same region(s) of the E2 component recognize the E1 and E3 components, to address the hypothesis that the loci of recognition of E2 for E1 or E3 are different. In the human complex, the issue to be addressed is determination of the regions of E2 component interacting with the E1 component, never accomplished before. Recent evidence indicates involvement of the kinase-dependent control of the human pyruvate dehydrogenase complex activity in both diabetes and cancer making structural studies of the human E2 component particularly timely. The studies proposed are intertwined with structural studies carried out in collaboration with highly productive and prominent groups.

描述（由申请人提供）：这是一个关于硫胺素二磷酸依赖酶的研究项目的更新申请，主要是关于大肠杆菌和人丙酮酸脱氢酶多酶复合物。这些复合物在几乎所有细胞的代谢过程中都起着关键作用，在进入克雷布斯循环（也称为三羧酸或柠檬酸循环）时，将糖酵解丙酮酸的产物转化为乙酰辅酶a。项目下一阶段的目标是：(1)确定酶结合的硫胺素相关中间体的限速步骤、电离状态和互变异构化，目的是将这些复合物的分离组分与4,600,000和10,000,000 Da复合物中的这些特性进行比较。在需要从头合成特定标记的硫胺素的创新中，PI建议使用固态核磁共振以及先进的溶液核磁共振方法来实现目标。(2)探索硫胺素酶活性中心环的迁移率与催化作用相关的假设。利用PI在2008年发表的方法，在这些研究中已经确定了大肠杆菌复合体E1和E2组分上的关键环，包括E2组分的脂酰结构域，它“访问”了所有三种组分的活性中心。(3)细菌和人类丙酮酸脱氢酶复合物组装的结构和功能后果的检查。在大肠杆菌复合体中，要解决的问题是E2组分的同一区域是否识别E1和E3组分，以解决E2对E1或E3的识别位点不同的假设。在人类复合体中，我们希望确定E2组分与E1组分相互作用的区域，这是以前从未完成的。PI使用的方法可以回答其他现有方法无法回答的问题，例如沿反应坐标确定辅酶4'-氨基嘧啶环上不同中间体上的质子位置，这是理解质子转移的关键问题。一群杰出的合作者已被招募，以获得在PI实验室无法获得的实验专业知识，其中包括因人类酶的专业知识而被选中的CoPI M. Patel，以及在一系列结构测定方面的长期合作者W. Furey。两个复合物的结果将使PI能够得出关于这种酶的整个超家族的一般结论，正如他最近发表的几篇文章所证明的那样。