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

Gonzalez-Nilo, FD; Krautwurst, H; Yevenes A.; Cardemil, E; Cachau, R

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 N? 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. ?G and ?H 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. © 2002 Elsevier Science B.V. All rights reserved.

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Título según WOS: Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase: theoretical and experimental study of the effect of glutamic acid 284 on the protonation state of lysine 213
Título según SCOPUS: Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase: Theoretical and experimental study of the effect of glutamic acid 284 on the protonation state of lysine 213
Título de la Revista: BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS
Volumen: 1599
Número: 01-feb
Editorial: Elsevier
Fecha de publicación: 2002
Página de inicio: 65
Página final: 71
Idioma: English
URL: http://linkinghub.elsevier.com/retrieve/pii/S1570963902004004
DOI:

10.1016/S1570-9639(02)00400-4

Notas: ISI, SCOPUS