INVESTIGATION OF PYRUVATE PHOSPHATE DIKINASE

丙酮酸磷酸二激酶的研究

• 批准号: 3289857
• 负责人: DEBRA DUNAWAY-MARIANO
• 金额: $ 11.72万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-01-01 至 1995-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3289857
• 关键词: Bacteroides; X ray crystallography; affinity labeling; bacterial proteins; chemical binding; computer assisted sequence analysis; computer simulation; conformation; divalent cations; electron spin resonance spectroscopy; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate complex; histidine; metal complex; metalloenzyme; molecular site; nuclear magnetic resonance spectroscopy; physical model; proteolysis; pyruvate kinase; rhodium; site directed mutagenesis; stop flow technique; thermodynamics

The overall goal of this project is to construct a detailed picture of (i) the active site regions where the three partial reactions comprising the PPDK reaction take place, (ii) the mechanisms by which these three partial reactions take place and (iii) the mechanism by which the putative histidine phosphoryl carrier shuttles from one active site region to the next. The specific aims of the project are: (1) To sequence the Bacteriodes symbiosus PPDK gene. (2) To crystallize PPDK and initiate X-ray crystallographic studies. (3) To determine the rate constants for the PPDK substrate binding and release steps and catalytic steps and to construct a free energy profile for the PPDK reaction. (4) To determine the stoichiometry, location and role of the metal ion cofactors during PPDK catalysis. (5) To identify the PPDK active site amino acid residues involved in substrate binding and catalysis. (6) To examine the structure of the active with regard to overlapping vs nonoverlapping sites for the three partial reactions and to test for movement of the putative "phosphoryl carrier histidine" between reaction sites during catalysis.

该项目的总体目标是构建 (i) 的详细图景 活性位点区域，其中三个部分反应组成 PPDK 反应发生，(ii) 这三个部分的机制 反应发生以及（iii）假定的机制 组氨酸磷酰基载体从一个活性位点区域穿梭至 下一个。该项目的具体目标是：（1）对拟杆菌进行测序 共生 PPDK 基因。 (2)使PPDK结晶并启动X射线 晶体学研究。 (3) 确定 PPDK 的速率常数 底物结合和释放步骤以及催化步骤并构建 PPDK 反应的自由能曲线。 (4) 确定 PPDK 过程中金属离子辅助因子的化学计量、位置和作用 催化。 (5) 鉴定PPDK活性位点氨基酸残基 参与底物结合和催化。 (6)检查结构 关于重叠与非重叠位点的活性 三个部分反应并测试假定的运动 催化过程中反应位点之间的“磷酰基载体组氨酸”。