MECHANISM OF E. COLI PYRUVATE DEHYDROGENASE COMPLEX-E1

描述（改编自申请人的摘要）：本研究的总体目标 研究是为了增强我们对结构-功能关系的理解， 包括丙酮酸的E1组分的机制和调节 脱氢酶多酶复合物分离自E.杆菌这种酶需要 硫胺素二磷酸（维生素B辅酶）和脱羧丙酮酸， 糖酵解的产物，引发一系列反应，主要产物是 乙酰辅酶A，柠檬酸循环的起始原料。中 所要求的时期的具体目标是：（1）生产野生型和 变体E1酶（变体是通过酶的定点诱变制备的）。 仅特异性携带编码E1的基因的质粒） 和结构研究，包括确定 与匹兹堡大学的William Furey合作（与 首席研究员合作解决了简单的酵母 丙酮酸脱羧酶结构）;（2）设计用于a.识别 负责催化作用的氨基酸，并赋予其在催化作用中的功能; B. 研究关键的硫胺素结合的中间体在酶上的命运; c.研究 催化反应中的限速步骤和催化反应中的过渡态结构 野生型和活性中心变体酶; d.确定的网站，并解决 底物氟丙酮酸使El失活的机制， 2-氧代-3-丁炔酸（在美国开发的有效底物类似物抑制剂） 首席研究员的实验室），一个假定的过渡态类似物，和 几种高抑制性单克隆抗体（也是在 主要研究者的实验室）;（3）确定研究中心的实验 在此，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， （4）将信息传输到活动中心; 首席研究员关于关键还原乙酰基的假设 E1和E2酶之间的转移。因为这种酶是一种关键的代谢酶 联合国，首席研究员认为，在未来几年，他和 他的合作者有机会为更好的分子水平 了解这一成员的一个非常大的类相关的多酶 配合物

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.