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

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The crystal structure of Escherichia coli phosphoenolpyruvate (PEP) carboxykinase shows Lys 213 is one of the ligands of enzyme-bound Mn 2+ [Nat. Struct. Biol. 4 (1997) 990]. The direct coordination of Mn 2+ by N ε of Lys 213 is only consistent with a neutral (uncharged) Lys 213, suggesting a low p K a for this residue. This work shows, through theoretical calculations and experimental analyses on homologous Saccharomyces cerevisiae PEP carboxykinase, how the microenvironment affects Mn 2+ binding and the protonation state of Lys 213. We show that Glu 284, a residue close to Lys 212, is required for correct protonation states of Lys 212 and Lys 213, and for Mn 2+ binding. Δ G and Δ H values for the proton reorganization processes were calculated to analyze the energetic stability of the two different protonation states of Lys 212 and Lys 213 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 Lys 212 must be protonated and Lys 213 neutral in the wild-type enzyme. On the other hand, the calculations on the Glu284Gln mutant suggest a more stable neutral Lys 212 and protonated Lys 213. Experimental measurements showed 3 orders of magnitude lower activity and a threefold increase in K m for Mn 2+ for Glu284Gln S. cerevisiae PEP carboxykinase when compared to wild type. The data here presented suggest that Glu 284 is required for Mn 2+ binding by S. cerevisiae PEP carboxykinase. We propose that Glu 284 modulates the p K a value of Lys 213 through electrostatic effects mediated by Lys 212.