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

The crystal structure of Escherichia coli phosphoenolpyruvate (PEP) carboxykinase shows Lys(213) is one of the ligands of enzyme-bound Mn2+ [Nat. Struct. Biol. 4 (1997) 990]. The direct coordination of Mn2+ by N-epsilon of Lys(213) is only consistent with a neutral (uncharged) Lys(213), suggesting a low pK(a) 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 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 Mn2(+) binding. DeltaG and DeltaH 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 Glu(284)Gln 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 Km for Mn2+ for Glu284Gln S. cerevisiae PEP carboxykinase when compared to wild type. The data here presented suggest that Glu(284) is required for Mn2+ binding by S. cerevisiae PEP carboxykinase. We propose that Glu(284) modulates the pK(a) value of Lys(213) through electrostatic effects mediated by Lys(212). (C) 2002 Elsevier Science B.V. All rights reserved.

See More