Journal
PROTEIN JOURNAL
Volume 26, Issue 2, Pages 135-141Publisher
SPRINGER
DOI: 10.1007/s10930-006-9054-z
Keywords
phosphoenolpyruvate carboxykinase; manganese binding site; Saccharomyces cererisiae; theoretical pK(a) calculations; manganese ligands in protein
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Funding
- NCRR NIH HHS [S10RR3790] Funding Source: Medline
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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 at. (1997) Nature Struct. Biol. 4, 990-994] shows that Lys(213) 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 epsilon-NH2 group of Lys(213). In this work, we evaluate the role of neighboring Phe(216) in contributing to provide a low polarity microenvironment suitable to keep the epsilon-NH2 of Lys(213) 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 p& of Lys. In agreement with this interpretation, theoretical calculations indicate an alkaline shift of 2.8 pH units in the pK(a) of the epsilon-amino group of Lys(213) upon Phe216Tyr mutation.
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