Abstract

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. Nature Struct. Biol. 4 (1997) 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 infrequent and suggests a low pKa value for the ε-NH2 group of Lys213. In this work, we evaluate the role of neighboring Phe416 in contributing to provide a low polarity microenvironment suitable to keep the ε-NH2 of Lys213 in the unprotonated form. Mutation Phe416Tyr 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. A study of the effect of pH on Km for Mn2+ indicate that the affinity of recombinant wild type enzyme for the metal ion is dependent on deprotonation of a group with pKa of 7.1 ± 0.2, compatible with the low pKa expected for Lys213. This pKa value increases at least 1.5 pH units upon Phe416Tyr mutation, in agreement with the expected effect of an increase in the polarity of Lys213 microenvironment. Theoretical calculations of the pKa of Lys213 indicate a value of 6.5 ± 0.9, and it increases to 8.2 ± 1.6 upon Phe416Tyr mutation. Additionally, mutation Phe416Tyr causes a loss of 1.3 kcal mol-1 in the affinity of the enzyme for PEP, an effect perhaps related to the close proximity of Phe416 to Arg70, a residue previously shown to be important for PEP binding. © 2006 Elsevier SAS. All rights reserved.