INVESTIGATION OF PYRUVATE PHOSPHATE DIKINASE

这个项目的总体目标是构建(I)的详细图景 活性中心区域，其中三个部分反应组成 PPDK反应的发生，(Ii)这三个部分 反应发生以及(Iii)推定的 组氨酸磷酸化载体从一个活性部位区域穿梭到 下一首。该项目的具体目标是：(1)对细菌进行测序 共生菌PPDK基因。(2)结晶PPDK，启动X射线 结晶学研究(3)测定PPDK的速率常数 底物结合和释放步骤和催化步骤，并构建 PPDK反应的自由能分布。(4)确定 PPDK中金属离子辅因子的化学计量、定位和作用 催化作用。(5)鉴定PPDK活性部位氨基酸残基 参与底物结合和催化作用。(6)检查结构 关于重叠站点与非重叠站点的活跃站点的 三个部分反应，并测试推定的运动 催化过程中反应部位之间的“磷酰化载体组氨酸”。

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.