Purification and functional analysis of the copper ATPase CopA of Enterococcus hirae.
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.
Wunderli-Ye
H
H
Department of Clinical Pharmacology, University of Berne, 3010 Berne, Switzerland.
Solioz
M
M
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D027682:Cation Transport Proteins; D003300:Copper / Q000378:metabolism; D000073840:Copper-Transporting ATPases; D016983:Enterococcus / Q000201:enzymology* / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
D002352:Carrier Proteins; D027682:Cation Transport Proteins; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D014638:Vanadates; D003300:Copper; D000251:Adenosine Triphosphatases; D000073840:Copper-Transporting ATPases; C000612460:copA protein, E coli