Abstract

Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase catalyzes the reversible formation of oxaloacetate and adenosine triphosphate from PEP, adenosine diphosphate and carbon dioxide, and uses Mn 2+ 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 Lys 213 is one of the ligands to Mn 2+ at the enzyme active site. Coordination of Mn 2+ to a lysyl residue is infrequent and suggests a low pK a value for the ?-NH 2 group of Lys 213. In this work, we evaluate the role of neighboring Phe 416 in contributing to provide a low polarity microenvironment suitable to keep the ?-NH 2 of Lys 213 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 Mn 2+, suggesting an increase of the pK a of Lys 213. A study of the effect of pH on K m for Mn 2+ indicate that the affinity of recombinant wild type enzyme for the metal ion is dependent on deprotonation of a group with pK a of 7.1 ± 0.2, compatible with the low pK a expected for Lys 213. This pK a value increases at least 1.5 pH units upon Phe416Tyr mutation, in agreement with the expected effect of an increase in the polarity of Lys 213 microenvironment. Theoretical calculations of the pK a of Lys 213 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 Phe 416 to Arg 70, a residue previously shown to be important for PEP binding. © 2006 Elsevier SAS. All rights reserved.