PRYUVATE DEHYDROGENASE E1: STRUCTURE-FUNCTION STUDIES

丙酮酸脱氢酶 E1：结构功能研究

• 批准号: 6387227
• 负责人: WILLIAM F FUREY
• 金额: $ 17.99万
• 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/6387227
• 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展我们对一大类对基本代谢至关重要的酶的结构与功能关系的了解；硫胺素二磷酸依赖性α-酮酸脱羧酶。 该项目重点研究在大型多酶复合物中发挥作用的硫胺素依赖性酶，以及它们与单独发挥作用的类似酶的关系。 尽管它们在生物化学中具有广泛的重要性并且经过多年的研究，但对于这种多酶复合物成分，仅存在一个最近获得的结构实例。 因此，即使是最基本的结构信息也刚刚可供该家族的任何成员使用，并且关于催化、调节和组装机制仍然存在许多问题。目标是首次对来自丙酮酸脱氢酶多酶复合物（来自大肠杆菌的 PDHc E1）的硫胺素依赖性 E1 成分进行高分辨率晶体结构分析，并将其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) 的结构，它在相同的底物上进行基本上相同的反应，但是孤立的而不是复合物，以及唯一有数据可用的其他 E1 结构；来自支链 2-氧代异戊酸脱氢酶多酶复合物的 E1b 组分。 后者 (BDHcE1b) 代表不同的 E1 类，与待研究的 E1 相比，没有序列同源性、不同的大小和亚基组成、不同的寡聚状态并且作用于不同的底物。 X 射线衍射方法还将用于提供有关由于与活化剂、底物和抑制剂相互作用而产生的结构变化的详细信息。 具体目标是：（1）确定和分析PDHc E1的结构，达到适合完整链追踪的分辨率； (2) 将完整的结构细化到尽可能高的分辨率； (3) 将PDHc E1结构与PDC的结构进行比较； (4)比较PDHc E1与BDHc E1b的结构； (5) 鉴定PDHc E1活性位点的氨基酸，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) 鉴定氟丙酮酸、2-氧代-3-丁炔酸、硫胺素-2-硫噻唑酮和单克隆抗体18A9的结合位点和抑制机制； (7) 确定GTP、乙酰辅酶A/辅酶A的调控结合位点以及信息传递至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。