Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1994 Apr 26;91(9):3593–3597. doi: 10.1073/pnas.91.9.3593

A recombinant bisphosphoglycerate mutase variant with acid phosphatase homology degrades 2,3-diphosphoglycerate.

M C Garel 1, N Arous 1, M C Calvin 1, C T Craescu 1, J Rosa 1, R Rosa 1
PMCID: PMC43626  PMID: 8170953

Abstract

To date no definite and undisputed treatment has been found for sickle cell anemia, which is characterized by polymerization of a deoxygenated hemoglobin mutant (HbS) giving rise to deformed erythrocytes and vasoocclusive complications. Since the erythrocyte glycerate 2,3-bisphosphate (2,3-DPG) has been shown to facilitate this polymerization, one therapeutic approach would be to decrease the intraerythrocytic level of 2,3-DPG by increasing the phosphatase activity of the bisphosphoglycerate mutase (BPGM; 3-phospho-D-glycerate 1,2-phosphomutase, EC 5.4.2.4). For this purpose, we have investigated the role of Gly-13, which is located in the active site sequence Arg9-His10-Gly11-Glu12-Gly13 in human BPGM. This sequence is similar to the Arg-His-Gly-Xaa-Arg* sequence of the distantly related acid phosphatases, which catalyze as BPGM similar phosphoryl transfers but to a greater extent. We hypothesized that the conserved Arg* residue in acid phosphatase sequences facilitates the phosphoryl transfer. Consequently, in human BPGM, we replaced by site-directed mutagenesis the corresponding amino acid residue Gly13 with an Arg or a Lys. In another experiment, we replaced Gly13 with Ser, the amino acid present at the corresponding position of the homologous yeast phosphoglycerate mutase (D-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1). Mutation of Gly13 to Ser did not modify the synthase activity, whereas the mutase and the phosphatase were 2-fold increased or decreased, respectively. However, replacing Gly13 with Arg enhanced phosphatase activity 28.6-fold, whereas synthase and mutase activities were 10-fold decreased. The presence of a Lys in position 13 gave rise to a smaller increase in phosphatase activity (6.5-fold) but an identical decrease in synthase and mutase activities. Taken together these results support the hypothesis that a positively charged amino acid residue in position 13, especially Arg, greatly activates the phosphoryl transfer to water. These results also provide elements for locating the conserved Arg* residue in the active site of acid phosphatases and facilitating the phosphoryl transfer. The implications for genetic therapy of sickle cell disease are discussed.

Full text

PDF
3593

Images in this article

3595Image
on p.3595
3596Image
on p.3596

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bazan J. F., Fletterick R. J., Pilkis S. J. Evolution of a bifunctional enzyme: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9642–9646. doi: 10.1073/pnas.86.24.9642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Calvin M. C., Blouquit Y., Garel M. C., Prehu M. O., Cohen-Solal M., Rosa J., Rosa R. Human bisphosphoglycerate mutase expressed in E coli: purification, characterization and structure studies. Biochimie. 1990 May;72(5):337–343. doi: 10.1016/0300-9084(90)90029-g. [DOI] [PubMed] [Google Scholar]
  3. Cherfils J., Rosa R., Garel M. C., Calvin M. C., Rosa J., Janin J. Crystallization and preliminary X-ray diffraction studies of the human erythrocyte bisphosphoglycerate mutase. J Mol Biol. 1991 Mar 20;218(2):269–270. doi: 10.1016/0022-2836(91)90710-n. [DOI] [PubMed] [Google Scholar]
  4. Craescu C. T., Schaad O., Garel M. C., Rosa R., Edelstein S. Structural modeling of the human erythrocyte bisphosphoglycerate mutase. Biochimie. 1992 Jun;74(6):519–526. doi: 10.1016/0300-9084(92)90149-9. [DOI] [PubMed] [Google Scholar]
  5. Dubart A., Romeo P. H., Tsapis A., Goossens M., Rosa R., Rosa J. Cell-free translation of messenger RNA for human bisphosphoglyceromutase. Biochem Biophys Res Commun. 1984 Apr 30;120(2):441–447. doi: 10.1016/0006-291x(84)91273-7. [DOI] [PubMed] [Google Scholar]
  6. Dykes G. W., Crepeau R. H., Edelstein S. J. Three-dimensional reconstruction of the 14-filament fibers of hemoglobin S. J Mol Biol. 1979 Jun 5;130(4):451–472. doi: 10.1016/0022-2836(79)90434-0. [DOI] [PubMed] [Google Scholar]
  7. Eaton W. A., Hofrichter J. Hemoglobin S gelation and sickle cell disease. Blood. 1987 Nov;70(5):1245–1266. [PubMed] [Google Scholar]
  8. Fothergill-Gilmore L. A., Watson H. C. The phosphoglycerate mutases. Adv Enzymol Relat Areas Mol Biol. 1989;62:227–313. doi: 10.1002/9780470123089.ch6. [DOI] [PubMed] [Google Scholar]
  9. Garel M. C., Joulin V., Le Boulch P., Calvin M. C., Préhu M. O., Arous N., Longin R., Rosa R., Rosa J., Cohen-Solal M. Human bisphosphoglycerate mutase. Expression in Escherichia coli and use of site-directed mutagenesis in the evaluation of the role of the carboxyl-terminal region in the enzymatic mechanism. J Biol Chem. 1989 Nov 15;264(32):18966–18972. [PubMed] [Google Scholar]
  10. Garel M. C., Lemarchandel V., Calvin M. C., Arous N., Craescu C. T., Prehu M. O., Rosa J., Rosa R. Amino acid residues involved in the catalytic site of human erythrocyte bisphosphoglycerate mutase. Functional consequences of substitutions of His10, His187 and Arg89. Eur J Biochem. 1993 Apr 1;213(1):493–500. doi: 10.1111/j.1432-1033.1993.tb17786.x. [DOI] [PubMed] [Google Scholar]
  11. Greaves D. R., Fraser P., Vidal M. A., Hedges M. J., Ropers D., Luzzatto L., Grosveld F. A transgenic mouse model of sickle cell disorder. Nature. 1990 Jan 11;343(6254):183–185. doi: 10.1038/343183a0. [DOI] [PubMed] [Google Scholar]
  12. INGRAM V. M. A specific chemical difference between the globins of normal human and sickle-cell anaemia haemoglobin. Nature. 1956 Oct 13;178(4537):792–794. doi: 10.1038/178792a0. [DOI] [PubMed] [Google Scholar]
  13. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Luzzatto L., Goodfellow P. Sickle cell anaemia. A simple disease with no cure. Nature. 1989 Jan 5;337(6202):17–18. doi: 10.1038/337017a0. [DOI] [PubMed] [Google Scholar]
  16. Pohlmann R., Krentler C., Schmidt B., Schröder W., Lorkowski G., Culley J., Mersmann G., Geier C., Waheed A., Gottschalk S. Human lysosomal acid phosphatase: cloning, expression and chromosomal assignment. EMBO J. 1988 Aug;7(8):2343–2350. doi: 10.1002/j.1460-2075.1988.tb03078.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Poillon W. N., Kim B. C. 2,3-Diphosphoglycerate and intracellular pH as interdependent determinants of the physiologic solubility of deoxyhemoglobin S. Blood. 1990 Sep 1;76(5):1028–1036. [PubMed] [Google Scholar]
  18. Rosa R., Audit I., Rosa J. Evidence for three enzymatic activities in one electrophoretic band of 3-phosphoglycerate mutase from red cells. Biochimie. 1975;57(9):1059–1063. doi: 10.1016/s0300-9084(75)80362-2. [DOI] [PubMed] [Google Scholar]
  19. Rosa R., Prehu M. O., Beuzard Y., Rosa J. The first case of a complete deficiency of diphosphoglycerate mutase in human erythrocytes. J Clin Invest. 1978 Nov;62(5):907–915. doi: 10.1172/JCI109218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Rosa R., Préhu M. O., Albrecht-Ellmer K., Calvin M. C. Partial characterization of the inactive mutant form of human red cell bisphosphoglyceromutase and comparison with an alkylated form. Biochim Biophys Acta. 1983 Jan 12;742(1):243–249. doi: 10.1016/0167-4838(83)90382-5. [DOI] [PubMed] [Google Scholar]
  21. Rose Z. B., Liebowitz J. 2,3-diphosphoglycerate phosphatase from human erythrocytes. General properties and activation by anions. J Biol Chem. 1970 Jun;245(12):3232–3241. [PubMed] [Google Scholar]
  22. Rubin E. M., Witkowska H. E., Spangler E., Curtin P., Lubin B. H., Mohandas N., Clift S. M. Hypoxia-induced in vivo sickling of transgenic mouse red cells. J Clin Invest. 1991 Feb;87(2):639–647. doi: 10.1172/JCI115041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ryan T. M., Townes T. M., Reilly M. P., Asakura T., Palmiter R. D., Brinster R. L., Behringer R. R. Human sickle hemoglobin in transgenic mice. Science. 1990 Feb 2;247(4942):566–568. doi: 10.1126/science.2154033. [DOI] [PubMed] [Google Scholar]
  24. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Taylor J. W., Ott J., Eckstein F. The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res. 1985 Dec 20;13(24):8765–8785. doi: 10.1093/nar/13.24.8765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Trudel M., Saadane N., Garel M. C., Bardakdjian-Michau J., Blouquit Y., Guerquin-Kern J. L., Rouyer-Fessard P., Vidaud D., Pachnis A., Roméo P. H. Towards a transgenic mouse model of sickle cell disease: hemoglobin SAD. EMBO J. 1991 Nov;10(11):3157–3165. doi: 10.1002/j.1460-2075.1991.tb04877.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Van Etten R. L., Davidson R., Stevis P. E., MacArthur H., Moore D. L. Covalent structure, disulfide bonding, and identification of reactive surface and active site residues of human prostatic acid phosphatase. J Biol Chem. 1991 Feb 5;266(4):2313–2319. [PubMed] [Google Scholar]
  28. Van Etten R. L. Human prostatic acid phosphatase: a histidine phosphatase. Ann N Y Acad Sci. 1982;390:27–51. doi: 10.1111/j.1749-6632.1982.tb40302.x. [DOI] [PubMed] [Google Scholar]
  29. White M. F., Fothergill-Gilmore L. A. Development of a mutagenesis, expression and purification system for yeast phosphoglycerate mutase. Investigation of the role of active-site His181. Eur J Biochem. 1992 Jul 15;207(2):709–714. doi: 10.1111/j.1432-1033.1992.tb17099.x. [DOI] [PubMed] [Google Scholar]
  30. Winn S. I., Watson H. C., Harkins R. N., Fothergill L. A. Structure and activity of phosphoglycerate mutase. Philos Trans R Soc Lond B Biol Sci. 1981 Jun 26;293(1063):121–130. doi: 10.1098/rstb.1981.0066. [DOI] [PubMed] [Google Scholar]
  31. Wishner B. C., Ward K. B., Lattman E. E., Love W. E. Crystal structure of sickle-cell deoxyhemoglobin at 5 A resolution. J Mol Biol. 1975 Oct 15;98(1):179–194. doi: 10.1016/s0022-2836(75)80108-2. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES