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

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.