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

DOI: 10.1007/s10930-006-9054-z

Cite this article as:
Yévenes, A., González-Nilo, F. & Cardemil, E. Protein J (2007) 26: 135. doi:10.1007/s10930-006-9054-z

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.


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



circular dichroism spectroscopy


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


high-performance liquid chromatography


molecular dynamics


3-(N-morpholino)propanesulfonic acid


oxaloacetic acid




sodium dodecylsulfate-polyacrylamide gel electrophoresis

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

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