The ATP hydrolytic activity of purified ZntA, a Pb(II)/Cd(II)/Zn(II)-translocating ATPase from Escherichia coli.
ZntA, a soft metal-translocating P1-type ATPase from Escherichia coli, confers resistance to Pb(II), Cd(II), and Zn(II). ZntA was expressed as a histidyl-tagged protein, solubilized from membranes with Triton X-100, and purified to homogeneity. The soft metal-dependent ATP hydrolysis activity of purified ZntA was characterized. The activity was specific for Pb(II), Cd(II), Zn(II), and Hg(II), with the highest activity obtained when the metals were present as thiolate complexes of cysteine or glutathione. The maximal ATPase activity of ZntA was approximately 3 micromol/(mg x min) obtained with the Pb(II)-thiolate complex. In the absence of thiolates, Cd(II) inhibits ZntA above pH 6, whereas the Cd(II)-thiolate complexes stimulate activity, suggesting that a metal-thiolate complex is the true substrate in vivo. These results are consistent with the physiological role of ZntA as mediator of resistance to toxic concentrations of the divalent soft metals, Pb(II), Cd(II), and Zn(II), by ATP-dependent efflux. Our results confirm that ZntA is the first Pb(II)-dependent ATPase discovered to date.
Sharma
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Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Rensing
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C
Rosen
B P
BP
Mitra
B
B
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000302:isolation & purification; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001692:Biological Transport; D002104:Cadmium / Q000378:metabolism; D003545:Cysteine / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007854:Lead / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry; D015032:Zinc / Q000378:metabolism
D008565:Membrane Proteins; D002104:Cadmium; D007854:Lead; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C109898:Zn(II)-translocating P-type ATPase; D015032:Zinc; D003545:Cysteine