Mechanisms of Enzyme Catalysis

描述(由申请人提供)：本项目的目标是确定PEP变位酶/异柠檬酸裂解酶和4-羟基苯甲酰辅酶A硫酯酶家族的选定成员的结构、功能和作用机制。这些信息将被用来将活性部位结构与催化联系起来，从而识别标记，这些标记可以应用于每个酶家族中所有未知蛋白质的功能分配。这些努力有望产生的新的蛋白质功能和代谢途径，以及活性部位结构的确定，将成为药物发现的基础。最后，从拟议的研究中，首席研究员和她的团队将深入了解参与这两个酶家族所代表的不同化学作用的酶的催化机制，以及这些催化机制如何从祖先的活性中心模板进化而来。具体目标1-4将阐述PEP变位酶/异柠檬酸裂解酶家族的四个成员的结构、功能和催化机制：洋葱伯克霍尔德氏菌的磷酸丙酮酸水解酶，铜绿假单胞菌的5，10-亚甲基四氢叶酸：3-甲基-2-氧丁酸羟甲基转移酶，与康乃馨花瓣死亡相关的蛋白，以及结核分枝杆菌中存在的功能未知的蛋白(Rv1998c)。具体的AIMS 5-9将涉及4-羟基苯甲酰辅酶A硫酯酶家族的五个成员的结构、功能和催化机制：4-羟基苯甲酰辅酶A硫酯酶、流感嗜血杆菌Tol-PAL途径的YgbC酶、大肠杆菌苯乙酸酯分解途径的P76084未知蛋白、嗜盐芽孢杆菌上龙胆酸盐途径的BH1 997未知蛋白和铜绿假单胞菌PA551 9与人类长链酰基辅酶A硫酯酶的同源物

DESCRIPTION (provided by applicant): The goal of this project is to determine structure, function, and mechanism of action of selected members of the PEP mutase/isocitrate lyase and 4-hydroxybenzoyl-CoA thioesterase enzyme families. This information will be used to relate active site structure to catalysis, and thereby identify markers, which can be applied in the assignment of function to all unknown proteins within each enzyme family. The novel protein functions and metabolic pathways that are anticipated to emerge from these efforts will, along with the active site structure determinations, serve as the foundation for drug discovery. Finally, from the proposed studies the principal investigator and her group will gain insight into the catalytic mechanisms of the enzymes mediating the diverse chemistries represented by the two enzyme families, and into how these catalytic mechanisms evolved from ancestral active site templates. Specific Aims 1-4 will address structure, function and catalytic mechanism in four members of the PEP mutase/ isocitrate lyase enzyme family: phosphonopyruvate hydrolase of Burkholderia cepacia, 5, 1 0-methylenetetrahydrofolate: 3-methyl-2-oxobutanoate hydroxymethyl transferase of Pseudomonas aeruginosa, a protein associated with petal death in carnation, and a protein of unknown function present in Mycobacterium tuberculosis (Rv1998c). Specific Aims 5-9 will address structure, function and catalytic mechanism in five members of the 4-hydroxybenzoyl-CoA thioesterase enzyme family: 4-hydroxybenzoyl-CoA thioesterase, the YgbC enzyme of the Haemophilus influenza Tol-pal Pathway, the P76084 unknown protein of the E.coli Phenylacetate Catabolic Pathway, the BH1 997 unknown protein of the Bacillus halodurans Upper Gentisate Pathway and the Pseudomonas aeruginosa PA551 9 homologue to the Human Long Chain Acyl-CoA Thioesterase