Structure and Mechanism of alpha-Ketoacid Decarboxylases and Dehydrogenases

描述（由申请人提供）：这是一个关于硫胺素二磷酸依赖酶的研究计划的更新申请，主要是关于E。大肠杆菌和人丙酮酸脱氢酶多酶复合物。这些复合物在几乎所有细胞的代谢中处于关键连接点，在进入克雷布斯循环（也称为三羧酸或柠檬酸循环）时将糖酵解产物葡糖酸转化为乙酰辅酶A。该项目下一阶段的目标是：（1）确定速率限制步骤，酶结合硫胺素相关中间体的电离和互变异构状态，目的是将复合物中分离组分的这些性质与4，600，000和10，000，000 Da复合物中的性质进行比较。在需要从头全合成特异性标记的噻托溴铵的创新中，PI建议使用固态NMR以及先进的溶液NMR方法来实现目标。(2)探索硫胺素酶活性中心环的流动性与催化作用相关的假说。利用PI在2008年发表的方法，E.大肠杆菌复合物的E1和E2组分已被确定用于这些研究，包括E2组分的硫辛酰结构域，其“访问”所有三个组分的活性中心。(3)在细菌和人丙酮酸脱氢酶复合物中组装的结构和功能后果的检查。在急诊大肠杆菌复合物中，要解决的问题是E2组分的相同区域是否识别E1和E3组分，以解决E2识别E1或E3的基因座不同的假设。在人类复合体中，我们希望确定E2组分与E1组分相互作用的区域，这是以前从未实现过的。PI使用的方法可以回答当前其他方法无法回答的问题，例如定义质子位置。在不同中间体处的辅酶的4 '-氨基嘧啶环上，沿着反应坐标，这是理解质子转移的关键问题。一批杰出的合作者已被招募的实验专业知识不提供在PI的实验室，其中M。帕特尔，一个CoPI选择的专业知识对人类酶，和W。Furey是一系列结构测定的长期合作者。从两个复合物的结果将使PI得出一般结论，对整个超家族的酶，已经证明了在他最近的几个出版物。 公共卫生相关性：这是一项关于二磷酸硫胺素依赖酶的研究计划的延期申请，主要是关于大肠杆菌。大肠杆菌和人丙酮酸脱氢酶多酶复合物。这些复合物在几乎所有细胞的代谢中处于关键连接点，在克雷布斯循环（也称为三羧酸或柠檬酸循环）的入口处将糖酵解产物葡糖酸转化为乙酰辅酶A。该项目下一阶段的目标包括：（1）确定速率限制步骤，酶结合硫胺素相关中间体的电离和互变异构状态，目的是将复合物中分离组分的这些性质与4，600，000和10，000，000 Da复合物中的性质进行比较。(2)探索硫胺素酶中活性中心环的流动性与催化作用相关的假设。(3)检查细菌和人丙酮酸脱氢酶复合物中组装的结构和功能后果。在急诊大肠杆菌复合物中，要解决的问题是E2组分的相同区域是否识别E1和E3组分，以解决E2识别E1或E3的基因座不同的假设。在人类复合体中，要解决的问题是确定E2组分与E1组分相互作用的区域，这是以前从未完成的。最近的证据表明，在糖尿病和癌症中，人类丙酮酸脱氢酶复合物活性的激酶依赖性控制的参与使得人类E2组分的结构研究特别及时。拟议的研究与结构研究交织在一起，结构研究是与富有成效的知名团体合作进行的。

DESCRIPTION (provided by applicant): This is a request for renewal of a research program on thiamin diphosphate-dependent enzymes, principally on the E. coli and human pyruvate dehydrogenase multienzyme complexes. These complexes are at a key junction in metabolism of virtually all cells, converting the product of glycolysis pyruvic acid to acetyl coenzyme A at the entry to the Krebs cycle (also known as the tricarboxylic acid or citric acid cycle). Goals for the next phase of the project are: (1) Determination of rate-limiting steps, states of ionization and tautomerization of enzyme-bound thiamin-related intermediates, with the intent to compare these properties in isolated components of the complexes to those in the 4,600,000 and 10,000,000 Da complexes. In innovation requiring de novo total synthesis of specifically labeled thiamins, the PI proposes to use solid state NMR, as well as advanced solution NMR methods to accomplish the goals. (2) Exploration of the hypothesis that the mobility of active center loops in thiamin enzymes is correlated with catalysis. With methods published by the PI in 2008, key loops on the E. coli complex's E1 and E2 components have been identified for these studies, including the lipoyl domain of the E2 component, which 'visits' the active centers of all three components. (3) Examination of the structural and functional consequences of assembly in the bacterial and human pyruvate dehydrogenase complexes. In the E. coli complex, the issue to be resolved is whether the same region(s) of the E2 component recognizes the E1 and E3 components, to address the hypothesis that the loci of recognition of E2 for E1 or E3 are different. In the human complex, we wish to determine the regions of E2 component interacting with the E1 component, never accomplished before The methods used by the PI could answer questions that no other current methodology can, such as the definition of proton positions on the 4'-aminopyrimidine ring of the coenzyme at distinct intermediates along the reaction coordinate, a key issue in understanding proton transfers. A group of outstanding collaborators have been recruited for experimental expertise not available in the PI's laboratory, among them M. Patel, a CoPI selected for expertise on the human enzyme, and W. Furey, a long-term collaborator in a string of structure determinations. Results from two complexes will enable the PI to draw general conclusions regarding the entire superfamily of such enzymes, as already demonstrated in several of his recent publications. PUBLIC HEALTH RELEVANCE: This is a request for renewal of a research program on thiamin diphosphate dependent enzymes, principally on the E. coli and human pyruvate dehydrogenase multienzyme complexes. These complexes are at a key junction in metabolism of virtually all cells, converting the product of glycolysis pyruvic acid to acetyl coenzyme A at the entry of the Krebs cycle (also known as the tricarboxylic acid or citric acid cycle). Goals for the next phase of the project include: (1) Determination of rate-limiting steps, states of ionization and tautomerization of enzyme-bound thiamin-related intermediates, with the intent to compare these properties in isolated components of the complexes to those in the 4,600,000 and 10,000,000 Da complexes. (2) Explore the hypothesis that the mobility of active center loops in thiamin enzymes is correlated with catalysis. (3) Examine the structural and functional consequences of assembly in the bacterial and human pyruvate dehydrogenase complexes. In the E. coli complex, the issue to be resolved is whether the same region(s) of the E2 component recognize the E1 and E3 components, to address the hypothesis that the loci of recognition of E2 for E1 or E3 are different. In the human complex, the issue to be addressed is determination of the regions of E2 component interacting with the E1 component, never accomplished before. Recent evidence indicates involvement of the kinase-dependent control of the human pyruvate dehydrogenase complex activity in both diabetes and cancer making structural studies of the human E2 component particularly timely. The studies proposed are intertwined with structural studies carried out in collaboration with highly productive and prominent groups.