Molecular analysis of an ATP-dependent anion pump.
The plasmid-borne arsenical resistance operon encodes an ATP-driven oxyanion pump for the extrusion of the oxyanions arsenite, antimonite and arsenate from bacterial cells. The catalytic component of the pump, the 63 kDa ArsA protein, hydrolyses ATP in the presence of its anionic substrate antimonite (SbO2-). The ATP analogue 5'-p-fluorosulphonylbenzoyladenosine was used to modify the ATP binding site(s) of the ArsA protein. From sequence analysis there are two potential nucleotide binding sites. Mutations were introduced into the N-terminal site. Purified mutant proteins were catalytically inactive and incapable of binding nucleotides. Conformational changes produced upon binding of substrates to the ArsA protein were investigated by measuring the effects of substrates on trypsin inactivation. The hydrophobic 45.5 kDa ArsB protein forms the membrane anchor for the ArsA protein. The presence of the ArsA protein on purified inner membrane can be detected immunologically. In the absence of the arsB gene no ArsA is found on the membrane. Synthesis of the ArsB protein is limiting for formation of the pump. Analysis of mRNA structure suggests a potential translational block to synthesis of the ArsB protein. Northern analysis of the ars message demonstrates rapid degradation of the mRNA in the arsB region.
Rosen
B P
BP
Department of Biochemistry, Wayne State University, School of Medicine, Detroit, Michigan 48201.
Hsu
C M
CM
Karkaria
C E
CE
Owolabi
J B
JB
Tisa
L S
LS
D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D053501:Arsenite Transporting ATPases; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D016623:Ion Pumps; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D009154:Mutation; D009690:Nucleic Acid Conformation; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics
D016623:Ion Pumps; D009097:Multienzyme Complexes; D012333:RNA, Messenger; D000251:Adenosine Triphosphatases; D053501:Arsenite Transporting ATPases