MECHANISM OF E. COLI PYRUVATE DEHYDROGENASE COMPLEX-E1

描述(改编自申请人的摘要)：这项工作的总体目标 研究是为了提高我们对结构-功能关系的理解， 包括丙酮酸电致发光成分的作用机制和调控 从大肠杆菌中分离的脱氢酶多酶复合体。这种酶需要 硫胺素二磷酸(维生素B辅酶)和脱羧酸丙酮酸， 糖酵解的产物，引发一系列反应，主要产物是 乙酰辅酶A，柠檬酸循环的起始物质。在这些人中 所要求期间的具体目标是：(1)野生型和 变异型EL酶(变异体是通过定点突变产生的 仅携带编码EL的基因的质粒) 和结构研究，包括确定一个X射线的结构 与匹兹堡大学的William Furey合作(与 首席研究员已经合作解决了更简单的酵母菌 丙酮酸脱羧酶结构)；(2)设计用于A.鉴定 负责催化的氨基酸，并分配在催化中的功能； 研究关键的硫胺素结合中间体在酶上的去向；C.研究 催化中的限速步骤和过渡态结构 野生型和活性中心变异酶；D.确定并解决 底物氟丙酮酸对EL的失活机理 2-氧代-3-丁酸(一种有效的底物类似物抑制剂 首席研究人员实验室)，一种假定的过渡态类似物，以及 几种高度抑制的单抗(也是在 首席调查员实验室)；(3)确定地点的实验(S) 在那里，各种代谢调节剂与EL相互作用，包括 辅因子和反应产物，以及GTP-和途径 我将信息传递给活动中心；以及(4)探索 主要研究者关于关键还原乙酰基的假说 EL和E2酶之间的转移。因为这种酶处于关键的代谢阶段 首席调查员相信，在接下来的几年里，他和 他的合作者有机会为更好的分子水平做出贡献 对一大类相关多酶中这一成员的理解 复合体。

DESCRIPTION (adapted from applicant's abstract): The overall objective of this research is to enhance our understanding of structure-function relationships, including the mechanism and regulation of the El component of the pyruvate dehydrogenase multienzyme complex isolated from E. coli. This enzyme requires thiamin diphosphate (the vitamin B coenzyme) and decarboxylates pyruvate, the product of glycolysis, to initiate a series of reactions, a major product being acetyl-Coenzyme A, the starting material for the citric acid cycle. Among the specific goals for the requested period are: (1) Production of wild-type and variant El enzymes (the variants being made by site directed mutagenesis of the plasmid specifically bearing only the gene coding for El) for both mechanistic and structural studies, including, determination of the X-ray structure in a collaboration wit William Furey, University of Pittsburgh (with whom the principal investigator has collaborated in the solution of the simpler yeast pyruvate decarboxylase structure); (2) Experiments designed to a. identify the amino acids responsible for catalysis, and assign the function in catalysis; b. study the fate of key thiamin-bound intermediates on the enzyme; c. study the rate-limiting steps in catalysis and the structure of transition states in wild-type and active center variant enzymes; d. identify the site of, and solve the mechanism of inactivation of El by substrate fluoropyruvate, 2-oxo-3-butynoic acid (a potent substrate analog inhibitor developed in the principal investigator's laboratory), a putative transition-state analog, and several highly inhibitory monoclonal antibodies (also developed in the principal investigator's laboratory); (3) Experiments to identify the site(s) at which a variety of metabolic regulators interact with the El, including the cofactors and the products of the reaction, as well as GTP- and the pathway by which the information i transmitted to the active center; and (4) Explore the principal investigator's hypothesis concerning the key reductive acetyl transfer between the El and E2 enzymes. Since this enzyme is at a key metabolic junction, the principal investigator believes that in the coming years he and his collaborators have an opportunity to contribute to a better molecular-level understanding of this member of a very large class of related multienzyme complexes.