PRYUVATE DEHYDROGENASE E1: STRUCTURE-FUNCTION STUDIES

丙酮酸脱氢酶 E1：结构功能研究

• 批准号: 6644169
• 负责人: WILLIAM F FUREY
• 金额: $ 17.87万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6644169
• 关键词: Escherichia coli; Pseudomonas; X ray crystallography; active sites; binding sites; enzyme complex; enzyme structure; enzyme substrate; guanosine triphosphate; monoclonal antibody; protein structure function; pyruvate decarboxylase; pyruvate dehydrogenase; thiamine pyrophosphate

The overall objective of this research is to extend our knowledge of structure-function relationships in a large class of enzymes of fundamental importance to basic metablism; the thiamin diphosphate dependent alpha-keto acid decarboxylases. The project focuses on thiamin dependent enzymes that function within large multienzyme complexes, and their relationship to similar enzymes functioning in isolation. Despite their widespread importance in biochemistry and many years of study, only a single, recently obtained structural example exists for such a multienzyme complex components. Thus even the most basic structural information has just become available for any member of this family, and numerous questions remain regarding the catalytic, regulatory and assembly mechanisms. The goal is to provide the first high-resolution crystal structure analysis of a thiamin dependent E1 component from a pyruvate dehydrogenase multi-enzyme complex, PDHc E1 from E. coli, and compare it to other related enzymes. The structures it's to be compared with include that for pyruvate decarboxylase (PDC), which carries out essentially the same reaction on the same substrate but in isolation rather than in a complex, and that for the only other E1 structure for which data are available; the E1b component from a branched chain 2-oxoisovalerate dehydrogenase multienzyme complex. The latter (BDHcE1b) represents a different E1 class having no sequence homology, a different size and subunit composition, a different oligomeric state and operates on a different substrate than the E1 to be studied. X-ray diffraction methods also will be used to provide detailed information about structural changes resulting from interaction wth activators, substrates and inhibitors. The specific aims are: (1) to determine and analyze the structure of PDHc E1 to a resolution suitable for a complete chain trace; (2) To refine the complete structure to the highest resolution possible; (3) To compare th PDHc E1 structure with that for PDC; (4) To compare the PDHc E1 structure with that for BDHc E1b; (5) To identify amino acids in the PDHc E1 active site, assign functional roles to each and identify residues involved in lipoamide binding; (6) To identify binding sites and inhibition mechanisms for fluropyruvate, 2-oxo-3-butynoic acid, thiamin-2-thiothiazolone and monoclonal antibody 18A9; (7) To identify regulatory binding sites for GTP, Acetyl-CoA/CoA and probe pathways for information transfer to catalytic sites; (8) To analyze structures of complexes with covalently bound reaction intermediate analogs (phosphonate or phosphinate analogs of pyruvate).

这项研究的总体目标是扩大我们的知识的结构-功能关系的一大类酶的基本重要性的基本代谢，硫胺素二磷酸依赖α-酮酸脱羧酶。 该项目的重点是在大型多酶复合物中发挥作用的硫胺素依赖酶，以及它们与孤立发挥作用的类似酶的关系。 尽管它们在生物化学中的广泛重要性和多年的研究，只有一个单一的，最近获得的结构的例子存在这样一个多酶复合物组分。 因此，即使是最基本的结构信息刚刚成为可用于该家庭的任何成员，和许多问题仍然关于催化，调节和组装机制。目的是提供第一个高分辨率的晶体结构分析的硫胺素依赖的E1组分从丙酮酸脱氢酶多酶复合物，PDHc E1从E。大肠杆菌中，并与其他相关酶进行比较。 与之比较的结构包括丙酮酸脱羧酶（PDC），它在相同的底物上进行基本相同的反应，但在隔离中而不是在复合物中，以及唯一的其他E1结构的数据可用;来自支链2-氧代异戊酸脱氢酶多酶复合物的E1 b组分。 后者（BDHcE 1b）代表一个不同的E1类，没有序列同源性，不同的大小和亚基组成，不同的寡聚状态，并在不同的基板上比E1进行研究。 X射线衍射方法也将用于提供有关与活化剂、底物和抑制剂相互作用引起的结构变化的详细信息。 具体目标是：（1）测定和分析PDHc E1的结构，使其达到适合于完整链迹的分辨率，（2）将完整结构细化到尽可能高的分辨率，（3）将PDHc E1的结构与PDC的结构进行比较，（4）将PDHc E1的结构与BDHc E1 b的结构进行比较，（5）将PDHc E1的结构与BDHc E1 b的结构进行比较，（6）将PDHc E1的结构与PDC的结构进行比较，（7）将PDHc E1的结构与BDHc E1 b的结构进行比较。（5）鉴定PDHc E1活性位点中的氨基酸，分配每个氨基酸的功能作用，并鉴定参与硫辛酰胺结合的残基;（6）确定氟丙酮酸、2-氧代-3-丁炔酸、硫胺素-2-硫代噻唑酮和单克隆抗体18 A9的结合位点和抑制机制;（7）鉴定GTP、乙酰辅酶A/辅酶A的调节结合位点，以及将信息传递到催化位点的探针途径;（8）分析与共价结合的反应中间体类似物（丙酮酸的膦酸盐或次膦酸盐类似物）的复合物的结构。