Purification and functional analysis of the copper ATPase CopA of Enterococcus hirae

  • Wunderli-Ye H
  • Solioz M
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Abstract

The Enterococcus hirae ATPase CopA is a member of the recently discovered heavy metal ATPases and shares 43% sequence identity with the human Menkes and Wilson copper ATPases. To study CopA biochemically, it was overexpressed in E. coli with an N-terminal histidine tag and purified to homogeneity by nickel affinity chromatography. The purified CopA catalyzed ATP hydrolysis with a Vmaxof 0.15 μmol/min/mg and a Kmfor ATP of 0.2 mM and had an optimum pH of 6.25. The activity was 3- to 4-fold stimulated by reconstitution into proteoliposomes. The enzyme formed an acylphosphate intermediate. Its kinetics of formation and the effects of inhibitors and metal ions upon it support a function of CopA in copper transport. Purification and functional reconstitution of CopA provides the basis to study copper transport in vitro. © 2001 Academic Press.

Author-supplied keywords

  • ATPase
  • Copper
  • Enterococcus hirae
  • Epitope tagging
  • Membrane protein
  • Phosphorylation
  • Proteoliposomes
  • Purification

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Authors

  • H. Wunderli-Ye

  • M. Solioz

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