Activation of Archaeoglobus fulgidus Cu+-ATPase CopA by cysteine

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Abstract

CopA, a thermophilic ATPase from Archaeoglobus fulgidus, drives the outward movement of Cu+ across the cell membrane. Millimolar concentration of Cys dramatically increases (≅ 800%) the activity of CopA and other PIB-type ATPases (Escherichia coli ZntA and Arabidopsis thaliana HMA2). The high affinity of CopA for metal (≅ 1 μM) together with the low Cu+-Cys KD (< 10- 10M) 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 Km suggesting no changes in the E1 ↔ E2 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. © 2006 Elsevier B.V. All rights reserved.

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Yang, Y., Mandal, A. K., Bredeston, L. M., Luis González-Flecha, F., & Argüello, J. M. (2007). Activation of Archaeoglobus fulgidus Cu+-ATPase CopA by cysteine. Biochimica et Biophysica Acta - Biomembranes, 1768(3), 495–501. https://doi.org/10.1016/j.bbamem.2006.09.013

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