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Regulation of Leishmania major PAS domain‐containing phosphoglycerate kinase by cofactor Mg 2+ ion at neutral pH
Author(s) -
Biswas Saroj,
Adhikari Ayan,
Mukherjee Aditi,
Das Sumit,
Adak Subrata
Publication year - 2020
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.15305
Subject(s) - phosphoglycerate kinase , isothermal titration calorimetry , chemistry , divalent , protonation , cofactor , enzyme , phosphoglycerate mutase , biochemistry , wild type , allosteric regulation , stereochemistry , mutant , biophysics , biology , glycolysis , ion , organic chemistry , gene
Recently, we described the PAS domain‐containing phosphoglycerate kinase (PGK) from Leishmania major (LmPAS‐PGK) that shows acidic pH (5.5)‐dependent optimum catalytic activity. The PAS domain of LmPAS‐PGK is expected to regulate PGK activity during catalysis, but the mechanism of regulation by PAS domain at the molecular level is uncharacterized. In this work, we have utilized the full‐length, PAS domain‐deleted, and mutant enzymes to measure the enzymatic activity in the presence of divalent cation at various pH values. Catalytic activity measurement indicates that Mg 2+ binding through PAS domain inhibits the PGK activity at pH 7.5, and this inhibition is withdrawn at pH 5.5. To identify the Mg 2+ binding residues of the PAS domain, we exploited a systematic mutational analysis of all (four) His residues in the PAS domain for potential divalent cation binding. Replacement of His‐57 with alanine resulted in depression in the presence of Mg 2+ at pH 7.5, but H71A, H89A, and H111A showed similar characteristics with respect to the wild‐type protein. Fluorescence and isothermal titration calorimetry studies revealed that H57 is responsible for Mg 2+ binding in the absence of substrates. Thus, the protonated form of His57 at acidic pH 5.5 destabilizes the Mg 2+ binding in the PAS domain, which is an essential requirement in the wild‐type LmPAS‐PGK for a conformational alteration in the sensor domain that, sequentially, activates the PGK domain, resulting in the synthesis of higher amounts of ATP.