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.
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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
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Acknowledgments
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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Yévenes, A., González-Nilo, F. & Cardemil, E. Relevance of phenylalanine 216 in the affinity of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase for Mn(II). Protein J 26, 135–141 (2007). https://doi.org/10.1007/s10930-006-9054-z
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DOI: https://doi.org/10.1007/s10930-006-9054-z