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, conf ers resistance to Pb(II), Cd(II), and Zn(II). ZntA was expressed as a histi dyl-tagged protein, solubilized from membranes with Triton X-100, and purif ied to homogeneity, The soft metal-dependent ATP hydrolysis activity of pur ified 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 wer e present as thiolate complexes of cysteine or glutathione. The maximal ATP ase activity of ZntA was similar to 3 mu mol/(mg.min) obtained with the Pb( II)-thiolate complex. In the absence of thiolates, Cd(II) inhibits ZntA abo ve pH 6, whereas the Cd(II)-thiolate complexes stimulate activity, suggesti ng that a metal-thiolate complex is the true substrate in vivo. These resul ts are consistent with the physiological role of ZntA as mediator of resist ance to toxic concentrations of the divalent soft metals, Pb(PI), Cd(II), a nd Zn(II), by ATP-dependent efflux. Our results confirm that ZntA is the fi rst Pb(II)-dependent ATPase discovered to date.