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1855805 
Journal Article 
Purification and functional analysis of the copper ATPase CopA of Enterococcus hirae 
Wunderli-Ye, H; Solioz, M 
2001 
Yes 
Biochemical and Biophysical Research Communications
ISSN: 0006-291X
EISSN: 1090-2104 
Elsevier 
280 
3 
713-719 
English 
11162579 
WOS:000166697300020 
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 V(max) of 0.15 micromol/min/mg and a K(m) for 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. 
copper; ATPase; purification; Enterococcus hirae; phosphorylation; membrane protein; epitope tagging; proteoliposomes