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).

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM037715-02
Application #
3293306
Study Section
Biochemistry Study Section (BIO)
Project Start
1986-12-01
Project End
1989-11-30
Budget Start
1987-12-01
Budget End
1988-11-30
Support Year
2
Fiscal Year
1988
Total Cost
Indirect Cost
Name
City of Hope/Beckman Research Institute
Department
Type
DUNS #
City
Duarte
State
CA
Country
United States
Zip Code
91010
Cheung, C W; Mas, M T (1996) Substrate-induced conformational changes in yeast 3-phosphoglycerate kinase monitored by fluorescence of single tryptophan probes. Protein Sci 5:1144-9
McPhillips, T M; Hsu, B T; Sherman, M A et al. (1996) Structure of the R65Q mutant of yeast 3-phosphoglycerate kinase complexed with Mg-AMP-PNP and 3-phospho-D-glycerate. Biochemistry 35:4118-27
Sherman, M A; Beechem, J M; Mas, M T (1995) Probing intradomain and interdomain conformational changes during equilibrium unfolding of phosphoglycerate kinase: fluorescence and circular dichroism study of tryptophan mutants. Biochemistry 34:13934-42
Mas, M T; Chen, H H; Aisaka, K et al. (1995) Effects of C-terminal deletions on the conformational state and denaturation of phosphoglycerate kinase. Biochemistry 34:7931-40
Sherman, M A; Fairbrother, W J; Mas, M T (1992) Characterization of the structure and properties of the His 62-->Ala and Arg 38-->Ala mutants of yeast phosphoglycerate kinase: an investigation of the catalytic and activatory sites by site-directed mutagenesis and NMR. Protein Sci 1:752-60
Sherman, M A; Dean, S A; Mathiowetz, A M et al. (1991) Site-directed mutations of arginine 65 at the periphery of the active site cleft of yeast 3-phosphoglycerate kinase enhance the catalytic activity and eliminate anion-dependent activation. Protein Eng 4:935-40
Sherman, M A; Szpikowska, B K; Dean, S A et al. (1990) Probing the role of arginines and histidines in the catalytic function and activation of yeast 3-phosphoglycerate kinase by site-directed mutagenesis. J Biol Chem 265:10659-65
Bailey, J M; Lin, L N; Brandts, J F et al. (1990) Substitution of a proline for alanine 183 in the hinge region of phosphoglycerate kinase: effects on catalysis, activation by sulfate, and thermal stability. J Protein Chem 9:59-67
Mas, M T; Bailey, J M; Resplandor, Z E (1988) Site-directed mutagenesis of histidine-388 in the hinge region of yeast 3-phosphoglycerate kinase: effects on catalytic activity and activation by sulfate. Biochemistry 27:1168-72
Mas, M T; Resplandor, Z E (1988) Structure-function relationships in 3-phosphoglycerate kinase: role of the carboxy-terminal peptide. Proteins 4:56-62

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