PRYUVATE DEHYDROGENASE E1: STRUCTURE-FUNCTION STUDIES

这项研究的总体目标是扩展我们对一大类对基本代谢具有重要意义的酶的结构-功能关系的认识；硫胺素二磷酸依赖性α -酮酸脱羧酶。该项目侧重于在大型多酶复合物中起作用的依赖硫胺素的酶，以及它们与单独起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，最近获得的结构例子存在于这种多酶复合物组分。因此，即使是最基本的结构信息，对于这个家族的任何成员都是可用的，关于催化，调节和组装机制仍然存在许多问题。目的是提供大肠杆菌丙酮酸脱氢酶多酶复合体PDHc E1中硫胺素依赖性E1组分的第一个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相同的反应，但是孤立的，而不是在络合物中，以及唯一可获得数据的其他E1结构；支链2-氧异戊酸脱氢酶多酶复合物中的E1b组分。后者（BDHcE1b）代表不同的E1类，与待研究的E1没有序列同源性，大小和亚基组成不同，寡聚态不同，作用于不同的底物。x射线衍射方法也将用于提供与活化剂、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)确定和分析PDHc E1的结构，以达到适合完整链迹的分辨率；(2)将整个结构细化到尽可能高的分辨率；(3)对比PDHc E1与PDC结构；(4)比较PDHc E1与BDHc E1b的结构；(5)鉴定PDHc E1活性位点的氨基酸，确定其功能作用，鉴定参与脂酰胺结合的残基；(6)确定氟丙酮酸、2-氧-3-丁酸、硫胺-2-硫代噻唑酮和单克隆抗体18A9的结合位点和抑制机制；(7)确定GTP、Acetyl-CoA/CoA的调控结合位点，探索信息传递到催化位点的途径；(8)分析共价结合反应中间类似物（膦酸盐或丙酮酸的膦酸类似物）的配合物的结构。

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).