title: Copper ATPase CopA from Escherichia coli: Quantitative Correlation between ATPase Activity and Vectorial Copper Transport.
abstract: Cu-ATPases are membrane copper transporters present in all kingdoms of life. They play a central role in Cu homeostasis by pumping Cu ions across cell membranes with energy derived from ATP hydrolysis. In this work, the Cu-ATPase CopA from Escherichia coli was expressed and purified in fully functional form and demonstrated to bind Cu(I) with subfemtomolar affinity. It was incorporated into the lipid membrane of giant unilamellar vesicles (GUVs) whose dimensions match those of eukaryotic cells. An1H NMR approach provided a quantitative ATPase activity assay for the enzyme either dissolved in detergent or embedded in GUV membranes. The activity varied with the Cu(I) availability in an optimized assay solution for either environment, demonstrating a direct correlation between ATPase activity and Cu(I) transport. Quantitative analysis of the Cu content trapped by the GUVs is consistent with a Cu:ATP turnover ratio of 1.
authors.affiliation: School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne , Parkville, Victoria 3010, Australia.
authors.forename: Chathuri J K
authors.initials: CJ
authors.lastname: Wijekoon
authors.affiliation: School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne , Parkville, Victoria 3010, Australia.
authors.forename: Saumya R
authors.initials: SR
authors.lastname: Udagedara
authors.affiliation: Max Planck Institute of Colloids and Interfaces , Science Park Golm, 14424 Potsdam, Germany.
authors.forename: Roland L
authors.initials: RL
authors.lastname: Knorr
authors.affiliation: Max Planck Institute of Colloids and Interfaces , Science Park Golm, 14424 Potsdam, Germany.
authors.forename: Rumiana
authors.initials: R
authors.lastname: Dimova
authors.affiliation: School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne , Parkville, Victoria 3010, Australia.
authors.forename: Anthony G
authors.initials: AG
authors.lastname: Wedd
authors.affiliation: School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne , Parkville, Victoria 3010, Australia.|The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville , Victoria 3010, Australia.
authors.forename: Zhiguang
authors.identifier: 0000-0001-6908-8897
authors.initials: Z
authors.lastname: Xiao
mesh_terms: D003300:Copper / Q000378:metabolism*; D000073840:Copper-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D017136:Ion Transport
chemical_list: D003300:Copper; D000073840:Copper-Transporting ATPases