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Biochemistry (Moscow)

, Volume 74, Issue 8, pp 910–916 | Cite as

Binding of ATP and its derivatives to selenophosphate synthetase from Escherichia coli

  • Y. V. PreabrazhenskayaEmail author
  • I. Y. Kim
  • T. C. Stadtman
Article

Abstract

Mechanistically similar selenophosphate synthetases (SPS) have been isolated from different organisms. SPS from Escherichia coli is an ATP-dependent enzyme with a C-terminal glycine-rich Walker sequence that has been assumed to take part in the first step of ATP binding. Three C-terminally truncated mutants of SPS, containing the N-terminal 238 (SPS238), 262 (SPS262), and 332 (SPS332) amino acids of the 348-amino-acid protein, have been extracted from cell pellets, and two of these (SPS262 and SPS332) have been purified to homogeneity. SPS238 has been obtained in a highly purified form. Binding of the fluorescent ATP-derivative TNP-ATP and Mn-ATP to the proteins was examined for all truncated mutants of SPS and a catalytically inactive C17S mutant. It has been shown that TNP-ATP can be used as a structural probe for ATP-binding sites of SPS. We observed two TNP-ATP binding sites per molecule of enzyme for wild-type SPS and SPS332 mutant and one TNP-ATP binding site for SPS238 mutant. The stoichiometry of Mn-ATP-binding was 2 mol of ATP per mol of protein determined with [14C]ATP by HPLC gel-filtration column chromatography under saturating conditions. The binding stoichiometries for SPS332, SPS262, and SPS238 were 2, 1.6, and 1, respectively. The C17S mutant exhibits about one third of wild type SPS TNP-ATP-binding ability and converts 12% of ATP in the ATPase reaction to ADP in the absence of selenide. The C-terminus contributes two thirds to the TNP-ATP binding; SPS238 likely has one ATP-binding site removed by truncation.

Key words

selenophosphate synthetase truncated mutants ATP-binding fluorescence enhancement 

Abbreviations

CHAPSO

3-[(3-cholamidopropyl)dimethylam-monio]-1-propanesulfonate oxide

Gu-HCl

guanidine hydrochloride

PIX

positional isotope exchange

SPS

selenophosphate synthetase

TNP-ATP

2′(3′)-O-(2,4,6-trini-trophenyl)adenosine 5′-triphosphate

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Supplementary material

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References

  1. 1.
    Veres, Z., Kim, I. Y., Scholz, T. D., and Stadtman, T. C. (1994) J. Biol. Chem., 269, 10597–10603.PubMedGoogle Scholar
  2. 2.
    Lacourciere, G. M., Mihara, H., Kurihara, T., Esaki, N., and Stadtman, T. C. (2000) J. Biol. Chem., 275, 23769–23773.PubMedCrossRefGoogle Scholar
  3. 3.
    Ehrenreich, A., Forschhammer, K., Tormay, P., Veprek, B., and Boeck, A. (1992) Eur. J. Biochem., 206, 767–773.PubMedCrossRefGoogle Scholar
  4. 4.
    Walker, H., Ferretti, J. A., and Stadtman, T. C. (1998) Proc. Natl. Acad. Sci. USA, 95, 2180–2185.PubMedCrossRefGoogle Scholar
  5. 5.
    Leinfelder, W., Forschhammer, K., Zinoni, F., Sawers, G., Mandrant-Berthelott, M.-A., and Boeck, A. (1988) J. Bacteriol., 170, 540–546.PubMedGoogle Scholar
  6. 6.
    Kim, I., Veres, Z., and Stadtman, T. C. (1992) J. Biol. Chem., 267, 19650–19654.PubMedGoogle Scholar
  7. 7.
    Leinfelder, W., Forschhammer, K., Veprek, B., Zehelein, E., and Boeck, A. (1990) Proc. Natl. Acad. Sci. USA, 87, 543–547.PubMedCrossRefGoogle Scholar
  8. 8.
    Lacourciere, G. M., and Stadtman, T. C. (1999) Proc. Natl. Acad. Sci. USA, 96, 44–48.PubMedCrossRefGoogle Scholar
  9. 9.
    Kubala, M., Plasek, J., and Amler, E. (2003) Eur. Biophys. J., 32, 363–369.PubMedCrossRefGoogle Scholar
  10. 10.
    Krepkiy, D., and Miziorko, H. M. (2005) Biochemistry, 44, 2671–2677.PubMedCrossRefGoogle Scholar
  11. 11.
    Wolfe, M. D. (2003) IUBMB Life, 55, 689–693.PubMedCrossRefGoogle Scholar
  12. 12.
    Patteson, K. G., Trivedi, N., and Stadtman, T. C. (2005) Proc. Natl. Acad. Sci. USA, 102, 12029–12034.PubMedCrossRefGoogle Scholar
  13. 13.
    Simonds, W., Koski, G., Streaty, R. A., Hjelmeland, L. M., and Klee, W. A. (1980) Proc. Natl. Acad. Sci. USA, 77, 4623–4627.PubMedCrossRefGoogle Scholar
  14. 14.
    Hiratsuka, T., and Uchida, K. (1973) Biochim. Biophys. Acta, 320, 635–647.PubMedGoogle Scholar
  15. 15.
    Kim, I. Y., and Stadtman, T. C. (1994) Proc. Natl. Acad. Sci. USA, 91, 7326–7329.PubMedCrossRefGoogle Scholar
  16. 16.
    Capieaux, E., Rapin, C., Thines, D., Dupon, Y., and Goffeau, A. (1993) J. Biol. Chem., 268, 21895–21900.PubMedGoogle Scholar
  17. 17.
    Ogasavara, Y., Lacourciere, G., Ishii, K., and Stadtman, T. C. (2005) Proc. Natl. Acad. Sci. USA, 102, 1012–1016.CrossRefGoogle Scholar
  18. 18.
    Low, S. C., Harney, J. W., and Berry, M. J. (1995) J. Biol. Chem., 270, 21659–21664.PubMedCrossRefGoogle Scholar
  19. 19.
    Mullins, L. S., Hong, S.-B., Gibson, G. E., Walker, H., Stadtman, T. C., and Raushel, F. M. (1997) J. Amer. Chem. Soc., 119, 6684–6685.CrossRefGoogle Scholar
  20. 20.
    Kim, I. Y., Veres, Z., and Stadtman, T. C. (1993) J. Biol. Chem., 268, 27020–27025.PubMedGoogle Scholar
  21. 21.
    Tamura, T., Yamamoto, S., Takahata, M., Sakaguchi, H., Tanaka, H., Stadtman, T. C., and Inagaki, K. (2004) Proc. Natl. Acad. Sci. USA, 101, 16162–16167.PubMedCrossRefGoogle Scholar
  22. 22.
    Martin, D. W., and Sachs, J. R. (2000) J. Biol. Chem., 275, 24512–24517.PubMedCrossRefGoogle Scholar
  23. 23.
    Ko, Y. H., Tomas, P. J., and Pedersen, P. L. (1994) J. Biol. Chem., 269, 14584–14588.PubMedGoogle Scholar
  24. 24.
    Soti, C., Vermes, A., Haystead, T., and Csermely, P. (2003) Eur. J. Biochem., 270, 2421–2428.PubMedCrossRefGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2009

Authors and Affiliations

  • Y. V. Preabrazhenskaya
    • 1
    • 2
    Email author
  • I. Y. Kim
    • 1
  • T. C. Stadtman
    • 1
  1. 1.Laboratory of Biochemistry, National Heart, Lung and Blood InstituteNational Institutes of HealthBethesdaUSA
  2. 2.Department of Biology and EcologyGrodno State UniversityGrodnoBelarus

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