Pyruvate Dehydrogenase E1: Structure-Function Studies

描述(申请人提供)：这项研究的总体目标是扩大我们对硫胺素二磷酸依赖的a-酮酸脱羧酶的结构-功能关系的知识，这是一大类普遍存在的酶，对细胞功能所需的新陈代谢及其相关的能量产生至关重要。这项研究的重点是在大型丙酮酸脱氢酶多酶复合体(PDHc)中工作的酶。与绝大多数生化反应途径不同的是，PDHc和其他多酶复合体通过底物在酶之间的简单扩散来运行，PDHc和其他多酶复合体在E1、E2和E3酶组分之间使用底物通道。这提供了一种获得非常高效率的方法，但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E.coliPDHc复合体的e1组分是结构上表达不足的细菌a2e1家族的成员，是二磷酸硫胺素依赖的，在整个酶反应中是速率决定的。它在序列上也与许多其他病原体中的对应物高度同源。广泛的、长期的目标是确定、分析和了解完整的PDHc复合体的结构和功能。目前的目标是利用和建立在前一个时期获得的来自E.coliPDHc的E1和E3组分的结构和生化信息。具体目的(1)是提供组装功能性多酶复合体所需的关键蛋白质-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。这将通过确定和分析由PDHc酶组分和/或其关键片段组成的二元络合物的晶体结构，并将它们与整体生物学功能相关联来实现。具体目的(2)是探索与以前发现的或被认为是稳定反应中间产物和可能的蛋白质-蛋白质组装所必需的构象变化相关的特征，并研究蛋白质-配体相互作用的影响。为了做到这一点，我们将确定和分析E1结构和相关的蛋白质-蛋白质复合体，在底物、底物类似物和新的催化位点定向抑制剂存在的情况下，以及引入一些突变。具体目标(3)是通过检查活性部位中单个残基突变引起的结构分支，以及沿着拟议的连接活性部位的“质子线”来探索机制问题。对于所有目的，分离的蛋白质、蛋白质-配体复合体或蛋白质-蛋白质复合体的X射线结晶学研究将与生化数据相结合，以获得该过程的完整图景。实现这些目标将有助于解决硫胺素依赖的酶催化方面的突出问题，并是朝着高分辨率分析整个4.57 x 106道尔顿3组分60亚基的长期目标迈出的下一步。与公共健康相关：硫胺素催化的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获得性或利用它的酶的异常都会造成严重的病理后果：例如脚气病、枫糖浆尿病、丙酮酸脱氢酶缺乏症(PDHA)相关的乳酸酸中毒、小头症、运动神经病、Leigh综合征以及包括阿尔茨海默氏症和帕金森氏症在内的神经系统疾病。拟议的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，并应提供可能有助于开发针对有效致病菌的抗菌药物的信息。

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.