Activation of Archaeoglobus fulgidus Cu+-ATPase CopA by cysteine

Abstract

CopA, a thermophilic ATPase from Archaeoglobus fulgidus, drives the outward movement of Cu(+) across the cell membrane. Millimolar concentration of Cys dramatically increases ( congruent with 800%) the activity of CopA and other P(IB)-type ATPases (Escherichia coli ZntA and Arabidopsis thaliana HMA2). The high affinity of CopA for metal ( congruent with 1 microM) together with the low Cu(+)-Cys K(D) (<10(-10)M) suggested a multifaceted interaction of Cys with CopA, perhaps acting as a substitute for the Cu(+) chaperone protein present in vivo. To explain the activation by the amino acid and further understand the mechanism of metal delivery to transport ATPases, Cys effects on the turnover and partial reactions of CopA were studied. 2-20 mM Cys accelerates enzyme turnover with little effect on CopA affinity for Cu(+), suggesting a metal independent activation. Furthermore, Cys activates the p-nitrophenyl phosphatase activity of CopA, even though this activity is metal independent. Cys accelerates enzyme phosphorylation and the forward dephosphorylation rates yielding higher steady state phosphoenzyme levels. The faster dephosphorylation would explain the higher enzyme turnover in the presence of Cys. The amino acid has no significant effect on low affinity ATP K(m) suggesting no changes in the E(1)<-->E(2) equilibrium. Characterization of Cu(+) transport into sealed vesicles indicates that Cys acts on the cytoplasmic side of the enzyme. However, the Cys activation of truncated CopA lacking the N-terminal metal binding domain (N-MBD) indicates that activation by Cys is independent of the regulatory N-MBD. These results suggest that Cys is a non-essential activator of CopA, interacting with the cytoplasmic side of the enzyme while this is in an E1 form. Interestingly, these effects also point out that Cu(+) can reach the cytoplasmic opening of the access path into the transmembrane transport sites either as a free metal or a Cu(+)-Cys complex.

Characterization of the P IB -Type ATPases Present in Thermus thermophilus

“…Based on metal stimulation of both the ATPase and pNPPase activities (Fig. 3), the current work shows that in vitro TtCopA favors Cu ϩ whereas TtCopB favors Cu 2ϩ , similar to the pattern observed with CopA and CopB from Archaeoglobus fulgidus (38,39,68), with the exception that the ATPase activity of TtCopA is not further stimulated by cysteine (5 to 20 mM) in the presence of 1 M Cu ϩ . By contrast, the CopB from Enterococcus hirae has been shown to act as an ATP-driven pump for Cu ϩ and Ag ϩ in inverted membrane vesicles (57).…”

Domain Organization and Movements in Heavy Metal Ion Pumps

“…S2). (31)(32)(33)(34). ATPase activity of CopA showed strong dependence on cysteine concentration with a maximum activity at 10 mM (Cu ϩ -ATPase) or 5 mM (Ag ϩ -ATPase) independent of the metal ion concentration (Fig.…”

Structure of the Two Transmembrane Cu+ Transport Sites of the Cu+-ATPases

“…p-Nitrophenyl Phosphatase Assays-p-Nitrophenyl phosphatase activity determinations were performed as described (32).…”