MECHANISM OF DOMAIN MOVEMENT IN PHOSPHOGLYCERATE KINASE

点击翻译按钮获取中文摘要

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).