Characterization of the catalytic subunit of an anion pump.
The ArsA protein, the 63-kDa catalytic subunit of an oxyanion-translocating ATPase, was purified by successive chromatography using Q-Sepharose, red agarose, and phenyl-Sepharose to a specific activity in excess of 1 mumol of ATP hydrolyzed per min per mg of protein. ATPase activity was dependent on the presence of the oxyanionic substrates. Inhibitors of other classes of ion-translocating ATPases had no effect on ArsA ATPase activity, including N,N'-dicyclohexyl-carbodiimide, azide, vanadate, and nitrate. The apparent Km for ATP was determined to be 0.13 mM. The optimal pH range for ATP hydrolysis was 7.5 to 7.8. ATPase activity required Mg2+ at a molar ratio of 2 ATP:1 Mg2+. Limited proteolysis by trypsin was used to study conformational changes produced upon binding of substrates to the ArsA protein. In the absence of substrates, the ArsA protein was rapidly cleaved by trypsin to a major product of 30 kDa. ATP was partially protected from trypsin digestion, while the anionic substrate antimonite alone had no effect on proteolysis. Combination of the two substrates nearly completely protected the ArsA protein from proteolysis. Proteolytic cleavage correlated with loss of anion-stimulated ATPase activity and substrate protection from cleavage correlated with retention of activity. These results demonstrate that ATP and antimonite together produce a conformational change which is different from that of the ArsA protein in the presence of either substrate alone and suggest interaction between the oxyanion and ATP binding sites.
Hsu
C M
CM
Department of Biochemistry, Wayne State University, School of Medicine, Detroit, Michigan 48201.
Rosen
B P
BP
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000838:Anions; D000965:Antimony / Q000494:pharmacology; D001151:Arsenic / Q000494:pharmacology; D053501:Arsenite Transporting ATPases; D018053:Arsenites; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002413:Cations, Divalent; D002845:Chromatography; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000032:analysis*; D016623:Ion Pumps; D008274:Magnesium / Q000494:pharmacology; D009097:Multienzyme Complexes; D011487:Protein Conformation; D013379:Substrate Specificity; D014357:Trypsin
D000838:Anions; D018053:Arsenites; D002352:Carrier Proteins; D002413:Cations, Divalent; D007473:Ion Channels; D016623:Ion Pumps; D009097:Multienzyme Complexes; C061374:antimonite; D000255:Adenosine Triphosphate; D000965:Antimony; D014357:Trypsin; D000251:Adenosine Triphosphatases; D053501:Arsenite Transporting ATPases; D008274:Magnesium; C015001:arsenite; D001151:Arsenic