Importance of aspartate 4 in the Mg2+dependent regulation of Leishmania major PAS domain-containing phosphoglycerate kinase

Magnesium is an important divalent cation for the regulation of catalytic activity. Recently, we have described that the Mg2+ binding through the PAS domain inhibits the phosphoglycerate kinase (PGK) activity in PAS domain-containing PGK from Leishmania major (LmPAS-PGK) at neutral pH 7.5, but PGK activity is derepressed at acidic pH 5.5. The acidic residue within the PAS domain of LmPAS-PGK is expected to bind the cofactor Mg2+ ion at neutral pH, but which specific acidic residue(s) is/are responsible for the Mg2+ binding is still unknown. To identify the residues, we exploited mutational studies of all acidic (twelve Asp/Glu) residues in the PAS domain for plausible Mg2+ binding. Mg2+ ion-dependent repression at pH 7.5 is withdrawn by substitution of Asp-4 with Ala, whereas other acidic residue mutants (D16A, D22A, D24A, D29A, D43A, D44A, D60A, D63A, D77A, D87A, and E107A) showed similar features compared to the wild-type protein. Fluorescence spectroscopic studies and isothermal titration calorimetry analysis showed that the Asp-4 is crucial for Mg2+ binding in the absence of both PGK's substrates. These results suggest that Asp-4 residue in the regulatory (PAS) domain of wild type enzymes is required for Mg2+ dependent repressed state of the catalytic PGK domain at neutral pH.

Automated summary

Magnesium is an important divalent cation for regulating enzyme activity. The binding of Mg2+ through the PAS domain inhibits phosphoglycerate kinase (PGK) activity in LmPAS-PGK at neutral pH, but PGK activity is derepressed at acidic pH. Mutation studies revealed that the Asp-4 residue is crucial for Mg2+ binding at neutral pH.