The Protein Journal

, Volume 26, Issue 2, pp 135–141 | Cite as

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

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

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 pK a 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 pK a of Lys213. In agreement with this interpretation, theoretical calculations indicate an alkaline shift of 2.8 pH units in the pK a 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



This work was supported by research grants FONDECYT 2010041 (AY) and 1030760 (EC). CD experiments were carried out at the Biophysics Instrumentation Facility of the University of Wisconsin-Madison, which was established by funding from NSF (BIR-9512577), NIH (S10RR3790), and the University of Wisconsin.


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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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