Substitution of a proline for alanine 183 in the hinge region of phosphoglycerate kinase: effects on catalysis, activation by sulfate, and thermal stability.
Substitution of a proline for alanine 183 in the hinge region of phosphoglycerate kinase: effects on catalysis, activation by sulfate, and thermal stability.
复制标题
磷酸甘油酸激酶铰链区中脯氨酸取代丙氨酸 183：对催化、硫酸盐活化和热稳定性的影响。
DOI：
10.1007/bf01024985
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
Mas,MT
中科院分区：
文献类型：
--
作者：
Bailey,JM;Lin,LN;Brandts,JF;Mas,MT
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. TheKmvalues 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, withTmnear 54°C, two transitions are evident for the mutant enzyme withTmvalues 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.
