Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase: Theoretical and experimental study of the effect of glutamic acid 284 on the protonation state of lysine 213

Fernando D. González-Nilo, Hans Krautwurst, Alejandro Yévenes, Emilio Cardemil, Raúl Cachau

Research output: Contribution to journalArticlepeer-review

8 Citations (Scopus)

Abstract

The crystal structure of Escherichia coli phosphoenolpyruvate (PEP) carboxykinase shows Lys213 is one of the ligands of enzyme-bound Mn2+ [Nat. Struct. Biol. 4 (1997) 990]. The direct coordination of Mn2+ by Nε of Lys213 is only consistent with a neutral (uncharged) Lys213, suggesting a low pKa for this residue. This work shows, through theoretical calculations and experimental analyses on homologous Saccharomyces cerevisiae PEP carboxykinase, how the microenvironment affects Mn2+ binding and the protonation state of Lys213. We show that Glu284, a residue close to Lys212, is required for correct protonation states of Lys 212 and Lys213, and for Mn2+ binding. ΔG and ΔH values for the proton reorganization processes were calculated to analyze the energetic stability of the two different protonation states of Lys212 and Lys213 in wild-type and Glu284Gln S. cerevisiae PEP carboxykinase. Calculations were done using two modeling approaches, ab-initio density functional calculations and free energy perturbation (FEP) calculations. Both methods suggest that Lys212 must be protonated and Lys213 neutral in the wild-type enzyme. On the other hand, the calculations on the Glu284Gln mutant suggest a more stable neutral Lys212 and protonated Lys213. Experimental measurements showed 3 orders of magnitude lower activity and a threefold increase in Km for Mn2+ for Glu284Gln S. cerevisiae PEP carboxykinase when compared to wild type. The data here presented suggest that Glu284 is required for Mn2+ binding by S. cerevisiae PEP carboxykinase. We propose that Glu284 modulates the pKa value of Lys213 through electrostatic effects mediated by Lys 212.

Original languageEnglish
Pages (from-to)65-71
Number of pages7
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1599
Issue number1-2
DOIs
Publication statusPublished - 23 Sep 2002

Keywords

  • Lysine pK
  • Phosphoenolpyruvate carboxykinase
  • Saccharomyces cerevisiae
  • Theoretical calculation

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biophysics
  • Biochemistry
  • Molecular Biology

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