ISSN:
1573-4943
Keywords:
3-Phosphoglycerate kinase
;
hinge-bending
;
domain movement
;
site-directed mutagenesis
;
thermal stability
Source:
Springer Online Journal Archives 1860-2000
Topics:
Chemistry and Pharmacology
Notes:
Abstract A “hinge-bending” domain movement has been postulated as an important part of the catalytic mechanism of phosphoglycerate kinase (PGK) (Bankset al., 1979). In order to test the role of the flexibility of a putative interdomain hinge in the substrate- and sulfate-induced conformational transitions, alanine-183 was replaced by proline using site-directed mutagenesis. The maximal velocity of the Ala 183→Pro mutant, measured at saturating concentrations of ATP and phosphoglycerate (5 mM and 10 mM, respectively) and in the absence of sulfate ions, is increased approximately 21% in comparison to the wild type PGK. TheK m values for both substrates are essentially unchanged. The effect of sulfate on the specific activity of the Ala 183→Pro mutant and the wild type PGK was measured in the presence of 1 mM ATP and 2 mM 3-phosphoglycerate (3-PG). A maximum activation of 70% was observed at 20 mM sulfate for the mutant enzyme, as compared to 130% activation at 30 mM sulfate for the wild type PGK. These results demonstrate that the increased rigidity of the putative hinge, introduced by the Ala→Pro mutation, does not impair catalytic efficiency of phosphoglycerate kinase, while it appears to decrease the sulfate-dependent activation. The differential scanning calorimetry (DSC) studies demonstrate an increased susceptibility of the Ala 183 → Pro mutant to thermal denaturation. In contrast to one asymmetric transition observed in the DSC scan for the wild type PGK, withT m near 54°C, two transitions are evident for the mutant enzyme withT m values of about 45 and 54°C. Using a thermodynamic model for two interacting domains, a decrease in the free energy of domain-domain interactions of about 2 kcal was estimated from the DSC data.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF01024985
