TY - JOUR
T1 - Functional properties of the copper-transporting ATPase ATP7B (the Wilson's disease protein) expressed in insect cells
AU - Tsivkovskii, Ruslan
AU - Eisses, John F.
AU - Kaplan, Jack H.
AU - Lutsenko, Svetlana
PY - 2002/1/11
Y1 - 2002/1/11
N2 - Copper-transporting ATPase ATP7B is essential for normal distribution of copper in human cells. Mutations in the ATP7B gene lead to copper accumulation in a number of tissues and to a severe multisystem disorder, known as Wilson's disease. Primary sequence analysis suggests that the copper-transporting ATPase ATP7B or the Wilson's disease protein (WNDP) belongs to the large family of cation-transporting P-type ATPases, however, the detailed characterization of its enzymatic properties has been lacking. Here, we developed a baculovirus-mediated expression system for WNDP, which permits direct and quantitative analysis of catalytic properties of this protein. Using this system, we provide experimental evidence that WNDP has functional properties characteristic of a P-type ATPase. It forms a phosphorylated intermediate, which is sensitive to hydroxylamine, basic pH, and treatments with ATP or ADP. ATP stimulates phosphorylation with an apparent Km of 0.95 ± 0.25 μM; ADP promotes dephosphorylation with an apparent Km of 3.2 ± 0.7 μM. Replacement of Asp1027 with Ala in a conserved sequence motif DKTG abolishes phosphorylation in agreement with the proposed role of this residue as an acceptor of phosphate during the catalytic cycle. Catalytic phosphorylation of WNDP is inhibited by the copper chelator bathocuproine; copper reactivates the bathocuproine-treated WNDP in a specific and cooperative fashion confirming that copper is required for formation of the acylphosphate intermediate. These studies establish the key catalytic properties of the ATP7B copper-transporting ATPase and provide a foundation for quantitative analysis of its function in normal and diseased cells.
AB - Copper-transporting ATPase ATP7B is essential for normal distribution of copper in human cells. Mutations in the ATP7B gene lead to copper accumulation in a number of tissues and to a severe multisystem disorder, known as Wilson's disease. Primary sequence analysis suggests that the copper-transporting ATPase ATP7B or the Wilson's disease protein (WNDP) belongs to the large family of cation-transporting P-type ATPases, however, the detailed characterization of its enzymatic properties has been lacking. Here, we developed a baculovirus-mediated expression system for WNDP, which permits direct and quantitative analysis of catalytic properties of this protein. Using this system, we provide experimental evidence that WNDP has functional properties characteristic of a P-type ATPase. It forms a phosphorylated intermediate, which is sensitive to hydroxylamine, basic pH, and treatments with ATP or ADP. ATP stimulates phosphorylation with an apparent Km of 0.95 ± 0.25 μM; ADP promotes dephosphorylation with an apparent Km of 3.2 ± 0.7 μM. Replacement of Asp1027 with Ala in a conserved sequence motif DKTG abolishes phosphorylation in agreement with the proposed role of this residue as an acceptor of phosphate during the catalytic cycle. Catalytic phosphorylation of WNDP is inhibited by the copper chelator bathocuproine; copper reactivates the bathocuproine-treated WNDP in a specific and cooperative fashion confirming that copper is required for formation of the acylphosphate intermediate. These studies establish the key catalytic properties of the ATP7B copper-transporting ATPase and provide a foundation for quantitative analysis of its function in normal and diseased cells.
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U2 - 10.1074/jbc.M109368200
DO - 10.1074/jbc.M109368200
M3 - Article
C2 - 11677246
AN - SCOPUS:0037059855
SN - 0021-9258
VL - 277
SP - 976
EP - 983
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 2
ER -