Abstract

Substrate-induced domain movement, which results in closure of the active site cleft, is an essential part of the catalytic mechanisms of 3- phosphoglycerate kinase.
The aim of propagation of ligand-induced conformational changes for this enzyme. The role of the side chain interactions at the interfaces between the adjacent alpha-helices, situated at the domain-domain interface, and their contacts with each domain will be examined using site directed mutagenesis. The location of the 3- phosphoglycerate binding site will also be determined using this approach. The effect of mutations on the catalytic properties of the mutants will be characterized. The structure and stability of mutants will be evaluated using biochemical and physical methods. These studies should contribute to a better understanding of the mechanisms of functionally-important conformational changes in proteins.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM037715-07
Application #
3293310
Study Section
Biochemistry Study Section (BIO)
Project Start
1986-12-01
Project End
1995-08-31
Budget Start
1993-09-01
Budget End
1994-08-31
Support Year
7
Fiscal Year
1993
Total Cost
Indirect Cost

  Institution

Name
City of Hope/Beckman Research Institute
Department
Type
DUNS #
City
Duarte
State
CA
Country
United States
Zip Code
91010

  Publications

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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