The Protein Journal

, Volume 26, Issue 2, pp 135–141

Relevance of phenylalanine 216 in the affinity of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase for Mn(II)

  • Alejandro Yévenes
  • Fernando D. González-Nilo
  • Emilio Cardemil
Article

Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase catalyzes the reversible formation of oxaloacetate and adenosine triphosphate from PEP, adenosine diphosphate and carbon dioxide, and uses Mn2+ as the activating metal ion. Comparison with the crystalline structure of homologous Escherichia coli PEP carboxykinase [Tari et al. (1997) Nature Struct. Biol.4, 990–994] shows that Lys213 is one of the ligands to Mn2+ at the enzyme active site. Coordination of Mn2+ to a lysyl residue is not common and suggests a low pKa value for the ε-NH2 group of Lys213. In this work, we evaluate the role of neighboring Phe216 in contributing to provide a low polarity microenvironment suitable to keep the ε-NH2 of Lys213 in the unprotonated form. Mutation Phe216Tyr shows that the introduction of a hydroxyl group in the lateral chain of the residue produces a substantial loss in the enzyme affinity for Mn2+, suggesting an increase of the pKa of Lys213. In agreement with this interpretation, theoretical calculations indicate an alkaline shift of 2.8 pH units in the pKa of the ε-amino group of Lys213 upon Phe216Tyr mutation.

Keywords

Phosphoenolpyruvate carboxykinase manganese binding site Saccharomyces cerevisiae theoretical pKa calculations manganese ligands in protein 

Abbreviations

CD

circular dichroism spectroscopy

HEPES

N-(2-hydroxyethyl)piperazine-N′-2(ethanesulfonic acid)

HPLC

high-performance liquid chromatography

MD

molecular dynamics

MOPS

3-(N-morpholino)propanesulfonic acid

OAA

oxaloacetic acid

PEP

phosphoenolpyruvate

SDS-PAGE

sodium dodecylsulfate-polyacrylamide gel electrophoresis

References

  1. Cotelesage J.J., Prasad L., Zeikus J.G., Laivenieks M., Delbaere L.T. (2005) Int. J. Biochem. Cell Biol. 37:1829–1837CrossRefGoogle Scholar
  2. Dunten P., Belunis C., Crowther R., Hollfelder K., Kammlott U., Levin W., Michel H., Ramsey G.B., Swain A., Weber D., Wertheimer S.J. (2002) J. Mol. Biol. 316:257–264CrossRefGoogle Scholar
  3. González-Nilo F.D., Krautwurst H., Yévenes A., Cardemil E., Cachau R. (2002) Biochim. Biophys. Acta 1599:65–71Google Scholar
  4. Holyoak T.,Sullivan S.M., Nowak T. (2006) Biochemistry 45:8254–8263CrossRefGoogle Scholar
  5. Jacob L.R., Vollert H., Rose M., Entian K.D., Bartunik L.J., Bartunik H. D. (1992) J. Chromatogr. 625:47–54CrossRefGoogle Scholar
  6. Krautwurst H., Encinas M.V., Marcus F., Latshaw S.P., Kemp R.G., Frey P.A., Cardemil E. (1995). Biochemistry 34:6382–6388CrossRefGoogle Scholar
  7. Krautwurst H., Bazaes S., González F.D., Jabalquinto A.M., Frey P.A., Cardemil E. (1998) Biochemistry 37:6295–6302CrossRefGoogle Scholar
  8. Krautwurst H., Roschzttardtz H., Bazaes S., Gonzalez-Nilo F.D., Nowak T., Cardemil E. (2002) Biochemistry 41:12763–12770CrossRefGoogle Scholar
  9. Lee M.H., Hebda C.A., Nowak T. (1981) J. Biol. Chem. 256:12793–12801Google Scholar
  10. Lee F.S., Chu Z.T., Warshel A. (1993) J. Comput. Chem. 14:161–185CrossRefGoogle Scholar
  11. Llanos L., Briones R., Yévenes A., González-Nilo F.D., Frey P.A., Cardemil E. (2001). FEBS Letters 493:1–5CrossRefGoogle Scholar
  12. Martel, A.E., and Smith, R.M. (1998). NIST standard references database 46, version 5.0Google Scholar
  13. Matte A, Tari L.W., Goldie H., Delbaere L.T.J. (1997) J. Biol. Chem. 272:8105–8108CrossRefGoogle Scholar
  14. Müller M., Müller H., Holzer H. (1981) J. Biol. Chem. 256:723–727Google Scholar
  15. Perella F.W. (1988) Anal. Biochem. 174:437–447CrossRefGoogle Scholar
  16. Sham Y.Y., Chu Z.T., Warshel A. (1997) J. Phys. Chem. 101:4458–4472Google Scholar
  17. Sugahara M., Ohshima N., Ukita Y., Sugahara M., Kunishima N. (2005) Acta Cryst. D61:1500–1507Google Scholar
  18. Tari L.W., Matte A., Goldie H., Delbaere L.T.J. (1997) Nature Struct. Biol. 4:990–994CrossRefGoogle Scholar
  19. Trapani S., Linss J., Goldenberg S., Fische H., Craievich A.F., Oliva G. (2001) J. Mol. Biol. 313:1059–1072CrossRefGoogle Scholar
  20. Utter M.F., Kolenbrander H.M. (1972) The Enzymes. 3rd, Academic Press, New York, 6:117–168Google Scholar
  21. Yévenes A., Espinoza R., Rivas-Pardo J.A., Villarreal J.M., González-Nilo F.D., Cardemil E. (2006) Biochimie 88:663–672CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2006

Authors and Affiliations

  • Alejandro Yévenes
    • 1
  • Fernando D. González-Nilo
    • 1
    • 2
  • Emilio Cardemil
    • 1
  1. 1.Departamento de Ciencias Químicas, Facultad de Química y BiologíaUniversidad de Santiago de ChileSantiago 33Chile
  2. 2.Centro de Bioinformática y Simulación MolecularUniversidad de TalcaTalcaChile

Personalised recommendations