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 Vmax of 0.15 μmol/min/mg and a Km 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. © 2001 Academic Press.
CITATION STYLE
Wunderli-Ye, H., & Solioz, M. (2001). Purification and functional analysis of the copper ATPase CopA of Enterococcus hirae. Biochemical and Biophysical Research Communications, 280(3), 713–719. https://doi.org/10.1006/bbrc.2000.4176
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