TRINITROPHENYL-ATP BINDING TO THE ARSA PROTEIN - THE CATALYTIC SUBUNIT OF AN ANION PUMP

The ars operon of the conjugative R-factor R773 confers resistance to arsenicals by coding for an anion pump for extrusion of arsenicals from cells of Escherichia coli. Extrusion of arsenite requires only two polypeptides, the ArsA and ArsB proteins. Purified ArsA protein exhibits oxyanion-stimulated ATPase activity and has been shown to bind ATP by photoaffinity labeling with [α-32P]ATP. From sequence analysis the ArsA protein is predicted to have two nucleotide binding folds, one in the N-terminal half and one in the C-terminal half of the protein. Purified ArsA protein bound a fluorescent ATP analogue, 2′,3′-O-(2,4,6-trinitrophenylcyclohexadienylidene)adenosine-5′-triphosphate, with an apparent stoichiometry of 2 mol of nucleotide per mole of ArsA. Strains expressing plasmids with mutations in the N-terminal consensus nucleotide sequence bound only 1 mol of nucleotide per mole of protein. © 1991.