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.
Similar content being viewed by others
References
Anderson, C. M., Zucker, F. H., and Steitz, T. A. (1979).Science 204, 375–380.
Banks, R. D., Blake, C. C. F., Evans, P. R., Haser, R., Rice, D. W., Hardy, G. W., Merrett, M., and Phillips, A. W. (1979).Nature 279, 773–777.
Bennett, W. S., Jr., and Steitz, T. A. (1978).Proc. Natl. Acad. Sci. USA 75, 4848–4852.
Blake, C. C. F., Rice, D. W., and Cohen, F. E. (1986).Int. J. Pept. Protein Res. 27, 443–448.
Bryant, T. N., Watson, H. C., and Wendell, P. L. (1974).Nature 247, 14–17.
Bücher, T. (1947).Biochim. Biophys. Acta 1, 292–314.
Chen, C. Y., Opperman, H., and Hitzeman, R. A. (1984).Nucleic Acids Res. 12, 8951–8970.
Edge, V., Allewell, N. M., and Sturtevant, J. M. (1985).Biochemistry 24, 5899–5906.
Hitzeman, R. A., Hagie, F. E., Hayflick, J. S., Chen, C. Y., Seeburg, P. H., and Derynck, R. (1982).Nucleic Acids Res. 10, 7791–7808.
Hu, C. Q., and Sturtevant, J. M. (1987).Biochemistry 26, 178–182.
Huber, R., and Bennett, W. S., Jr. (1983).Biopolymers 22, 261–279.
Janin, J., and Wodak, S. J. (1983).Prog. Biophys. Mol. Biol. 42, 21–78.
Khamis, M. M., and Larsson-Raznikiewicz, M. (1981).Biochim. Biophys. Acta 657, 190–194.
Kunkel, T. A. (1985).Proc. Natl. Acad. Sci. USA 82, 488–492.
Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987).Methods Enzymol. 154, 367–382.
Laemmli, U. K. (1970).Nature 227, 680–685.
Larsson-Raźnikiewicz, M. (1967).Biochim. Biophys. Acta 132, 33–40.
Larsson-Raźnikiewicz, M. (1973).Arch. Biochem. Biophys. 158, 754–762.
Larsson-Raźnikiewicz, M., and Arvidsson, L. (1971).Eur. J. Biochem. 22, 506–512.
Larsson-Raźnikiewicz, M., and Jansson, J. R. (1973).FEBS Lett. 29, 345–347.
Lesk, A. M., and Chothia, C. (1984).J. Mol. Biol. 174, 175–191.
Manly, S. P., Matthews, K. S., and Sturtevant, J. M. (1985).Biochemistry 24, 3842–3846.
Mas, M. T., Bailey, J. M., and Resplandor, Z. E. (1988).Biochemistry 27, 1168–1172.
Mas, M. T., Chen, C. Y., Hitzeman, R. A., and Riggs, A. D. (1986).Science 233, 788–790.
Mas, M. T., and Resplandor, Z. E. (1988).Proteins, Structure, Function, and Genetics 4, 56–62.
Mas, M. T., Resplandor, Z. E., and Riggs, A. D. (1987).Biochemistry 26, 5369–5377.
Matthews, B. W. (1987).Biochemistry 26, 6885–6888.
Mori, N., Singer-Sam, J., and Riggs, A. D. (1986).FEBS Lett. 204, 313–317.
Pickover, C. A., McKay, D. B., Engelman, D. M., and Steitz, T. A. (1979).J. Biol. Chem. 254, 11323–11329.
Roustan, C., Fattoum, A., Jeanneau, R., and Pradel, L.-A. (1980).Biochemistry 19, 5168–5175.
Schierbeck, B., and Larsson-Raźnikiewicz, M. (1979).Biochim. Biophys. Acta 568, 195–204.
Schulz, G. E., and Schirmer, R. H. (1978). InPrinciples of Protein Structure. Springer-Verlag, New York, pp 17–26.
Scopes, R. K. (1973).Enzymes (3rd Ed)8, 335–351.
Scopes, R. K. (1978a).Eur. J. Biochem. 85, 503–516.
Scopes, R. K. (1978b).Eur. J. Biochem. 91, 119–129.
Sturtevant, J. M. (1987).Ann. Rev. Phys. Chem. 38, 463–488.
Tanswell, P., Westhead, E. W., and Williams, R. J. P. (1976).Eur. J. Biochem. 63, 249–262.
Watson, H. C., Walker, N. P. C., Shaw, P. J., Bryant, P. L., Fothergill, L. A., Perkins, R. E., Conroy, S. C., Dobson, M. J., Tuite, M. F., Kingsman, A. J., and Kingsman, S. M. (1982).EMBO J. 1, 1635–1640.
Wilson, C. A. B., Hardman, N., Fothergill-Gilmore, L. A., Gamblin, S. J., and Watson, H. C. (1987).Biochem. J. 241, 609–614.
Wilson, H. R., Williams, R. J. P., Littlechild, J. A., and Watson, H. C. (1988).Eur. J. Biochem. 170, 529–538.
Zagursky, R. J., Baumeister, K., Lomax, N., and Berman, M. L. (1985).Gene Anal. Tech. 2, 89–94.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Bailey, J.M., Lin, LN., Brandts, J.F. et al. 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 (1990). https://doi.org/10.1007/BF01024985
Received:
Published:
Issue Date:
DOI: https://doi.org/10.1007/BF01024985