Pyruvate Dehydrogenase E1: Structure-Function Studies

描述(申请人提供)：这项研究的总体目标是扩大我们对硫胺素二磷酸依赖的α-酮酸脱羧酶的结构-功能关系的知识；硫胺素二磷酸依赖的α-酮酸脱羧酶是一大类普遍存在的酶，对基础代谢具有基本重要性。该项目的重点是在大型多酶复合体中运行的依赖于硫胺素的二磷酸酶，特别是丙酮酸脱氢酶多酶复合体(PDHc)的E1部分。这种多酶复合体使用底物通道作为获得高效率的手段，关于关键的所需相互作用的细节知之甚少。来自E.ColiPDHc复合体的靶E1成分是细菌α(2)E1家族的成员，并且在序列上与许多病原体中的对应成分高度同源。广泛的长期目标是确定、分析和了解完整的PDHc复合体的结构和功能，而短期目标是探索基本的E1反应及其与E2组分的相互作用。具体目的是：(1)分析含有PDHc E1酶的蛋白质-配体复合体以及抑制剂、底物和/或效应物。这包括催化位点定向的抑制剂硫胺硫代噻唑酮二磷酸(ThTTDP)、乙酰基膦酸甲酯、2-氧代-3-丁酸和氟丙酮酸；底物/受体-底物类似物丙酮酸、硫辛酸和epsilon-N-脂酰赖氨酸(脂酰胺)；以及效应剂鸟苷三磷酸(GTP)、乙酰辅酶A和NAD/NADH。(2)获得大肠杆菌apo-E1酶和PDHc多酶复合体中其他(非E1)酶组分的结构-功能信息，并确定复杂组装/功能所需的蛋白质-蛋白质相互作用的位置。(3)确定大肠杆菌PDHc E1中关键催化位点的单残基突变对结构的影响。(4)扩展PDHc相互作用的结构-功能分析，以包括来自其他来源的相关酶，特别是来自结核分枝杆菌的PDHc复合体。采用的主要方法包括分离蛋白质、蛋白质-配基复合体和蛋白质-蛋白质复合体的X射线结晶学研究。

DESCRIPTION (provided by applicant): The overall objective of this research is to extend our knowledge of structure-function relationships in thiamin diphosphate dependent alpha-keto acid decarboxylases; a large and ubiquitous class of enzymes of fundamental importance to basic metabolism. The project focuses on thiamin diphosphate dependent enzymes thal operate within large multienzyme complexes, and in particular, the E1 component of the pyruvate dehydrogenase multienzyme complex (PDHc). Such multienzyme complexes employ substrate channeling as a means of obtaining high efficiency, and few details are known regarding the key required interactions. The target E1 component from the E. coli PDHc complex is a member of the bacterial alpha (2) E1 family, and is highly homologous in sequence with its counterparts in many pathogenic organisms. The broad, long-term objective is to determine, analyze and understand the structure and function of an intact PDHc complex, while the short term objective is to probe the basic E1 reaction and its interactions with the E2 component. The specific aims are: (1) To analyze protein-ligand complexes containing the PDHc E1 enzyme along with inhibitors, substrates and/or effectors. This includes the catalytic site directed inhibitors thiaminthiothiazolone diphosphate (ThTTDP), methyl acetylphosphinate, 2-oxo-3-butynoic acid, and fluropyruvate; the substrate/acceptor-substrate analogs pyruvate, lipoic acid and epsilon-N-Lipoyllysine (lipoamide); and the effectors guanosine triphosphate (GTP), acetyI-CoAJCoA and NAD+/NADH. (2) To obtain structure-function information for the E. coli apo-E1 enzyme and for other (non-E1) enzymatic components in the PDHc multienzyme complex, and to determine sites of protein-protein interaction required for complex assembly/function. (3) To identify structural effects resulting from single residue mutations in E. coli PDHc E1 at key catalytic site locations. (4) to extend structure-function analysis of PDHc interactions to include related enzymes from other sources, in particular, the PDHc complex from mycobacterium tuberculosis. The major method to be employed involves x-ray crystallographic studies of isolated proteins, protein-ligand complexes and protein-protein complexes.