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.