PRYUVATE DEHYDROGENASE E1: STRUCTURE-FUNCTION STUDIES

这项研究的总体目标是扩展我们对基础代谢至关重要的一大类酶的结构-功能关系的知识，这些酶是依赖于硫胺素二磷酸的α-酮酸脱羧酶。该项目的重点是在大型多酶复合体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具有广泛的重要性和多年的研究，但对于这种多酶复杂成分来说，只有一个最近获得的结构例子。因此，即使是最基本的结构信息也只是对这个家族的任何成员都是可用的，关于催化、调节和组装机制的许多问题仍然存在。目的是首次对来自大肠杆菌的丙酮酸脱氢酶多酶复合体PDHc E1中依赖硫胺素的E1组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。要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，但是在隔离而不是在复合体中进行的，以及唯一可以获得数据的其他E1结构的结构；来自支链2-氧异戊酸脱氢酶多酶复合体的E1b成分。后者(BDHcE1b)代表不同的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，并在与要研究的E1不同的底物上工作。X射线衍射法还将用于提供与激活剂、底物和缓蚀剂相互作用引起的结构变化的详细信息。具体目的是：(1)确定和分析PDHc E1的结构，使其达到适合全链示踪的分辨率；(2)将完整的结构提纯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 E1b的结构进行比较；(5)鉴定PDHc E1活性部位中的氨基酸，赋予每个氨基酸功能角色，并鉴定参与脂胺结合的残基；(6)确定氟丙酮酸、2-氧代-3-丁酸、硫胺素-2-硫代噻唑酮和单抗18A9的结合部位和抑制机理；(7)鉴定GTP、乙酰辅酶A/辅酶A的调节结合部位，探索信息传递到催化部位的途径；(8)分析共价结合反应中间类似物(丙酮酸的膦或膦类似物)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).