Pyruvate Dehydrogenase E1: Structure-Function Studies

丙酮酸脱氢酶 E1：结构功能研究

• 批准号: 7647511
• 负责人: WILLIAM F FUREY
• 金额: $ 29.32万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7647511
• 关键词: Acids; Active Sites; Affect; Alzheimer' s Disease; Anti-Bacterial Agents; Beriberi; Binding; Biochemical; Biochemical Reaction; Biological; Biological Process; Carboxy-Lyases; Catalysis; Catalytic Domain; Cell physiology; Communication; Complex; Coupled; DNA Sequence Rearrangement; Data; Development; Diffusion; Diphosphates; Disease; Enzymes; Escherichia coli; Family; Goals; Hand; Keto Acids; Knowledge; Lactic Acidosis; Leigh Disease; Ligands; Location; Maple Syrup Urine Disease; Metabolism; Microcephaly; Motion; Motor; Multienzyme Complexes; Mutation; N-terminal; Neuropathy; Organism; Parkinson Disease; Pathway interactions; Peripheral; Point Mutation; Process; Production; Proteins; Protons; Pyruvate; Pyruvate Dehydrogenase E1; Pyruvates; Reaction; Research; Resolution; Role; Site; Solutions; Structure; Structure-Activity Relationship; Thiamine; Thiamine Pyrophosphate; Thioctic Acid; Variant; Work; acetylphosphinate; analog; dalton; dimer; enzyme structure; inhibitor/antagonist; lipoamide; lipoyllysine; member; mutant; nervous system disorder; pathogenic bacteria; protein complex; protein protein interaction; public health relevance; pyruvate dehydrogenase; pyruvate dehydrogenase multienzyme complex E1

DESCRIPTION (provided by applicant): The overall objective of this research is to extend our knowledge of structure-function relationships in thiamine diphosphate dependent a-keto acid decarboxylases, a large and ubiquitous class of enzymes of critical importance to metabolism and its associated energy production required for cellular function. The research focuses on enzymes that operate within the large, pyruvate dehydrogenase multienzyme complex (PDHc). Unlike the vast majority of biochemical reaction pathways that operate through simple diffusion of substrates and products between enzymes, PDHc and other multienzyme complexes employ substrate channeling between the E1, E2, and E3 enzymatic components. This provides a means of obtaining very high efficiency, and many key details are lacking regarding the required intramolecular interactions and processes. The E1 component from the E. coli PDHc complex is a member of the structurally underrepresented bacterial a2 E1 family, is thiamin diphosphate dependent, and is rate determining in the overall enzymatic reaction. It is also highly homologous in sequence with its counterparts in many other pathogenic organisms. The broad, long-term objective is to determine, analyze and understand the structure and function of an intact PDHc complex. The immediate objective is to exploit and build upon the structural and biochemical information obtained in the previous period for the E1 and E3 components from E. coli PDHc. Specific aim (1) is to provide detailed information about key protein-protein interactions necessary to assemble the functional multienzyme complex, and about the substrate channeling mechanism used to transfer products/substrates between enzymatic components within it. This will be achieved by determining and analyzing crystal structures of binary complexes made up from PDHc enzymatic components and/or their key fragments, and correlating them with the overall biological function. Specific aim (2) is to probe features associated with conformational changes previously found or thought to be necessary for stabilization of reaction intermediates and possibly protein-protein assembly, and to study the effects of protein-ligand interactions. To do this we will determine and analyze E1 structures and the associated protein-protein complexes in the presence of substrate, substrate analogs, and a new catalytic site directed inhibitor, as well as with some mutations introduced. Specific aim (3) is to probe mechanistic issues by examining structural ramifications arising from single residue mutations both in the active site, and along a proposed "proton wire" connecting active sites. For all aims x-ray crystallographic studies of isolated proteins, protein-ligand complexes, or protein-protein complexes will be coupled with biochemical data to obtain a complete picture of the process. Achieving these aims will help resolve the outstanding issues in thiamin-dependent enzymatic catalysis, and is the next step towards the long range goal of high resolution analysis of the entire 4.57 x 106 Dalton, 3 component 60 subunit containing PDHc complex. PUBLIC HEALTH RELEVANCE: The importance of thiamin catalyzed reactions has long been recognized, since abnormalities in either the availability of the vitamin B1 derived thiamin diphosphate or in the enzymes that utilize it have severe pathological consequences: for example, beri-beri, maple syrup urine disease, Pyruvate Dehydrogenase Deficiency (PDHA) associated lactic acidosis, microcephaly, motor neuropathy, Leigh syndrome, and neurological diseases including Alzheimer's and Parkinson's. The proposed work will advance our understanding of the precise mechanisms in key enzymatic pathways utilizing thiamin to provide energy for cellular functions, and should provide information likely to be useful in development of antibacterial agents that specifically target potent, pathogenic bacteria.

描述（由申请人提供）：本研究的总体目标是扩展我们对硫胺素二磷酸依赖的a-酮酸脱羧酶的结构-功能关系的认识，这是一大类普遍存在的酶，对细胞功能所需的代谢及其相关能量产生至关重要。该研究的重点是在大型丙酮酸脱氢酶多酶复合物（PDHc）内运作的酶。与绝大多数通过底物和产物在酶之间的简单扩散进行的生化反应途径不同，PDHc和其他多酶复合物在E1、E2和E3酶组分之间使用底物通道。这为获得非常高的效率提供了一种手段，并且缺乏关于所需的分子内相互作用和过程的许多关键细节。来自大肠杆菌PDHc复合体的E1组分是结构上代表性不足的细菌a2 E1家族的成员，依赖于硫胺二磷酸，并且在整个酶促反应中决定速率。它在序列上也与许多其他病原生物中的对应物高度同源。广泛的，长期的目标是确定，分析和理解完整的PDHc复合物的结构和功能。当前的目标是利用和建立在前一阶段从大肠杆菌PDHc中获得的E1和E3成分的结构和生化信息。具体目标(1)是提供组装功能性多酶复合物所需的关键蛋白质-蛋白质相互作用的详细信息，以及用于在其中的酶组分之间转移产物/底物的底物通道机制。这将通过测定和分析由PDHc酶组分和/或其关键片段组成的二元配合物的晶体结构，并将其与整体生物学功能相关联来实现。具体目标(2)是探索与构象变化相关的特征，这些特征先前发现或被认为是稳定反应中间体和可能的蛋白质-蛋白质组装所必需的，并研究蛋白质-配体相互作用的影响。为了做到这一点，我们将在底物、底物类似物、新的催化位点定向抑制剂以及引入一些突变的情况下，确定和分析E1结构和相关的蛋白质-蛋白质复合物。具体目的(3)是通过检查活性位点和连接活性位点的“质子线”中单个残基突变所产生的结构分支来探索机制问题。对于所有目标，分离蛋白、蛋白配体复合物或蛋白-蛋白复合物的x射线晶体学研究将与生化数据相结合，以获得整个过程的完整图像。实现这些目标将有助于解决硫胺素依赖性酶催化的突出问题，并且是实现整个4.57 x 106道尔顿，3组分60亚基含PDHc复合物的高分辨率分析的长期目标的下一步。公共卫生相关性：硫胺素催化反应的重要性早已被认识到，因为维生素B1衍生的硫胺素二磷酸的可用性或利用它的酶的异常会导致严重的病理后果：例如，脚气病、枫糖尿病、丙酮酸脱氢酶缺乏症（PDHA）相关的乳酸酸中毒、小头畸形、运动神经病变、Leigh综合征和包括阿尔茨海默病和帕金森病在内的神经系统疾病。这项工作将促进我们对利用硫胺素为细胞功能提供能量的关键酶途径的精确机制的理解，并可能为开发专门针对强效致病菌的抗菌剂提供有用的信息。