MECHANISM OF DOMAIN MOVEMENT IN PHOSPHOGLYCERATE KINASE

拟议研究的目的是审查结构基础 磷酸甘油酸盐中底物诱导的结构域运动的机制 激酶（PGK）。 提出了一种实验方法， 基因工程和物理化学方法相结合。 PGK 由通过铰链连接的两个球状结构域组成。 的作用 二级结构元件和单个氨基酸位于 PGK的铰链区，以及表面环，将使用 抗坏血酸定向诱变。 氨基酸取代和/或 删除将介绍，基于计算机图形分析酵母 PGK。 构象变化的离域程度将是 通过在离铰链不同距离处引入突变来评估。 的 底物结合事件和相关结构域之间的偶联 运动将通过研究这些突变对 构象、柔性、催化活性和配体结合性质 突变酶的基因 引入的突变对 还将评价稳定性和折叠性能。 结构和 突变磷酸甘油酸激酶的功能将使用 物理化学方法（CD，荧光，UV光谱，化学 修饰和动力学）。

The objective of the proposed research is to examine the structural basis for the mechanism of substrate-induced domain movement in phosphoglycerate kinase (PGK). An experimental approach is proposed which involves a combination of genetic engineering and physico-chemical methods. PGK consists of two globular domains which are connected by a hinge. The role of the secondary structure elements and individual amino acids situated in the hinge region of PGK, and of the surface loops, will be studied using oligonucleotide-directed mutagenesis. Amino acid substitutions and/or deletions will be introduced, based on computer graphics analysis of yeast PGK. The extent of delocalization of the conformational changes will be assessed by introducing mutations at various distances from the hinge. The coupling between the substrate binding event and the relative domain movement will be examined by studying the effect of these mutations on conformation, flexibility, catalytic activity and ligand binding properties of the mutant enzymes. The effects of the introduced mutations on the stability and folding properties will also be evaluated. Structure and function of the mutant phosphoglycerate kinases will be studied using physico-chemical methods (CD, fluorescence, UV spectroscopy, chemical modifications and kinetics).