INTERACTION OF SUBSTRATE AND EFFECTOR-BINDING SITES IN THE ARSA ATPASE

The ars operon of plasmid R773 confers resistance to antimonials and a rsenicals in Escherichia coli by encoding an ATP-dependent extrusion s ystem for the oxyanions, The catalytic subunit, the ArsA protein, is a n ATPase with two nucleotide binding consensus sequences, one in the N -terninal half and one in the C-terminal half of the protein. The ArsA ATPase is allosterically activated by tricoordinate binding of As(3+) or Sb(3+) to three cysteine thiolates. Previous measurements suggeste d that the intrinsic fluorescence of tryptophans might be useful for e xamining binding of Mg(2+)ATP and antimonite. In the present study an increase in intrinsic tryptophan fluorescence was observed upon additi on of Mg(2+)ATP. This enhancement was reversed by addition of antimoni te. The ArsA protein contains four tryptophan residues: Trp159, Trp253 , Trp522, and Trp524. The first two were altered to tyrosine residues by site-directed mutagenesis. Cells expressing both the arsA(W159Y) an d arsA(W253Y) mutations retained resistance to arsenite, and the purif ied W159Y and W253Y proteins retained ATPase activity. While the intri nsic tryptophan fluorescence of the W253Y protein responded to additio n of Mg(2+)ATP, intrinsic tryptophan fluorescence in the purified W159 Y protein was no longer enhanced by substrate. These results suggest t hat Trp159 is conformationally coupled to one or both of the nucleotid e binding sites and provides a useful probe for the interaction of eff ector and substrate binding sites.