Functional properties of the copper-transporting ATPase ATP7B (the Wilson's disease protein) expressed in insect cells

J Biol Chem. 2002 Jan 11;277(2):976-83. doi: 10.1074/jbc.M109368200. Epub 2001 Oct 24.


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 K(m) of 0.95 +/- 0.25 microm; ADP promotes dephosphorylation with an apparent K(m) of 3.2 +/- 0.7 microm. Replacement of Asp(1027) 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Diphosphate / metabolism
  • Adenosine Triphosphatases / genetics
  • Adenosine Triphosphatases / metabolism*
  • Adenosine Triphosphate / metabolism
  • Animals
  • Baculoviridae / genetics
  • Baculoviridae / metabolism
  • Cation Transport Proteins / genetics
  • Cation Transport Proteins / metabolism*
  • Cell Fractionation
  • Cell Line
  • Chelating Agents / pharmacology
  • Copper / metabolism*
  • Copper-Transporting ATPases
  • Free Radical Scavengers / pharmacology
  • Humans
  • Indicators and Reagents / metabolism
  • Phenanthrolines / pharmacology
  • Phosphines / metabolism
  • Phosphorylation
  • Protein Structure, Secondary
  • Recombinant Proteins / metabolism
  • Spodoptera / cytology
  • Spodoptera / metabolism


  • Cation Transport Proteins
  • Chelating Agents
  • Free Radical Scavengers
  • Indicators and Reagents
  • Phenanthrolines
  • Phosphines
  • Recombinant Proteins
  • tris(2-carboxyethyl)phosphine
  • Adenosine Diphosphate
  • Copper
  • Adenosine Triphosphate
  • bathocuproine
  • Adenosine Triphosphatases
  • ATP7B protein, human
  • Copper-Transporting ATPases