Pyruvate Dehydrogenase E1: Structure-Function Studies

描述（由申请人提供）：本研究的总体目标是扩展我们对二磷酸硫胺素依赖性α-酮酸脱羧酶结构-功能关系的了解，这是一种对代谢及其相关能量产生至关重要的大而普遍存在的酶，细胞功能所需。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。与绝大多数通过酶之间底物和产物的简单扩散操作的生化反应途径不同，PDHc和其他多酶复合物在E1，E2和E3酶组分之间采用底物通道。这提供了获得非常高效率的手段，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。E.大肠杆菌PDHc复合物是结构上代表性不足的细菌α 2 E1家族的成员，是二磷酸硫胺素依赖性的，并且在整个酶促反应中是速率决定性的。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，分析和了解完整的PDHc复合物的结构和功能。当前的目标是利用和建立在前一时期从E. coli PDHc.具体目的（1）是提供关于组装功能性多酶复合物所必需的关键蛋白质-蛋白质相互作用的详细信息，以及关于用于在其中的酶组分之间转移产物/底物的底物通道机制的详细信息。这将通过测定和分析由PDH c酶组分和/或其关键片段组成的二元复合物的晶体结构来实现，并将它们与整体生物功能联系起来。具体目标（2）是探索与先前发现或认为是反应中间体稳定和可能的蛋白质-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。要做到这一点，我们将确定和分析E1结构和相关的蛋白质-蛋白质复合物的存在下，底物，底物类似物，和一个新的催化位点定向抑制剂，以及一些突变的介绍。具体目标（3）是通过检查活性位点和沿着连接活性位点的提议的“质子线”的单残基突变产生的结构分支来探测机制问题。对于所有的目标，分离的蛋白质，蛋白质-配体复合物，或蛋白质-蛋白质复合物的X射线晶体学研究将与生物化学数据相结合，以获得该过程的完整图像。实现这些目标将有助于解决硫胺素依赖性酶催化中的突出问题，并且是实现整个4.57 × 106道尔顿，3组分60亚基的含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具有严重的病理后果：例如脚气病、枫糖浆尿病、丙酮酸脱氢酶缺乏（PDHA）相关的乳酸酸中毒、小头畸形、运动神经病、Leigh综合征，和神经系统疾病包括阿尔茨海默氏症和帕金森氏症。拟议的工作将推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专门针对强效致病菌的抗菌剂的信息。

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.