Reaction of the ArsA adenosinetriphosphatase with 2-(4'-maleimidoanilino)naphthalene-6-sulfonic acid.
The oxyanion-translocating ATPase encoded by the plasmid-borne ars operon catalyzes extrusion of antimonials and arsenicals from cells of Escherichia coli, thus providing resistance to those toxic oxyanions. The purified catalytic subunit of the ATPase, the ArsA protein, exhibits oxyanion-stimulated ATPase activity. The nature of the oxyanion binding site was probed by reaction with the fluorescent sulfhydryl probe 2-(4'-maleimidoanilino)naphthalene-6-sulfonic acid (MIANS). Our results indicate that MIANS reacts with the ArsA protein in an antimonite-dependent manner. After the protein had been modified with MIANS, two of four cysteines in the ArsA protein had reacted with the probe in the absence of the oxyanionic substrate, and three in the presence of antimonite. The quantum yield of the MIANS-ArsA protein adduct was significantly higher if modification of the protein had occurred in the presence of oxyanionic substrates. Thus binding of the anionic substrate of the pump produces a conformational change in the ArsA protein such that a single additional cysteinyl residue reacts more readily with the sulfhydryl probe.
Ksenzenko
M Y
MY
Department of Biochemistry, Wayne State University, School of Medicine, Detroit, Michigan 48201.
Kessel
D H
DH
Rosen
B P
BP
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism* / Q000494:pharmacology; D000838:Anions / Q000378:metabolism; D053501:Arsenite Transporting ATPases; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D016623:Ion Pumps; D009097:Multienzyme Complexes; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011815:R Factors; D012997:Solvents; D013050:Spectrometry, Fluorescence; D013057:Spectrum Analysis
D000817:Anilino Naphthalenesulfonates; D000838:Anions; D016623:Ion Pumps; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D012997:Solvents; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; D004228:Dithionitrobenzoic Acid; D000251:Adenosine Triphosphatases; D053501:Arsenite Transporting ATPases