Asp(45) is a Mg2+ ligand in the ArsA ATPase

The ATPase activity of ArsA, the catalytic subunit of the plasmid-encoded, ATP-dependent extrusion pump for arsenicals and antimonials in Escherichia coli, is allosterically activated by arsenite or antimonite. Magnesium is e ssential for ATPase activity. To examine the role of Asp(45) mutants were c onstructed in which Asp(45) was changed to Glu, Asn, or Ale. Cells expressi ng these mutated arsA genes lost arsenite resistance to varying degrees. Pu rified D45A and D45N enzymes were inactive, The purified D45E enzyme exhibi ted approximately 5% of the wild type activity with about a 5-fold decrease in affinity for Mg2+. Intrinsic tryptophan fluorescence was used to probe Mg2+ binding. ArsA containing only Trp(159) exhibited fluorescence enhancem ent upon the addition of MgATP, which was absent in D45N and D45A. As anoth er measure of conformation, limited trypsin digestion was used to estimate the surface accessibility of residues in ArsA. ATP and Sb(III) synergistica lly protected wild type ArsA from trypsin digestion. Subsequent addition of Mg2+ increased trypsin sensitivity. D45N and D45A remained protected by AT P and Sb(III) but lost the Mg2+ effect. D45E exhibited an intermediate Mg2 response. These results indicate that Asp(45) is a Mg2+-responsive residue , consistent with its function as a Mg2+ ligand.