The regulation of catalytic activity of the menkes copper-translocating P-type ATPase. Role of high affinity copper-binding sites.
The Menkes protein is a transmembrane copper translocating P-type ATPase. Mutations in the Menkes gene that affect the function of the Menkes protein may cause Menkes disease in humans, which is associated with severe systemic copper deficiency. The catalytic mechanism of the Menkes protein, including the formation of transient acylphosphate, is poorly understood. We transfected and overexpressed wild-type and targeted mutant Menkes protein in yeast and investigated its transient acyl phosphorylation. We demonstrated that the Menkes protein is transiently phosphorylated by ATP in a copper-specific and copper-dependent manner and appears to undergo conformational changes in accordance with the classical P-type ATPase model. Our data suggest that the catalytic cycle of the Menkes protein begins with the binding of copper to high affinity binding sites in the transmembrane channel, followed by ATP binding and transient phosphorylation. We propose that putative copper-binding sites at the N-terminal domain of the Menkes protein are important as sensors of low concentrations of copper but are not essential for the overall catalytic activity.
Voskoboinik
I
I
Department of Genetics, The University of Melbourne, Parkville 3010, Australia.
Mar
J
J
Strausak
D
D
Camakaris
J
J
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry*; D002384:Catalysis; D027682:Cation Transport Proteins; D002462:Cell Membrane; D003300:Copper / Q000378:metabolism*; D000073840:Copper-Transporting ATPases; D004305:Dose-Response Relationship, Drug; D005816:Genetic Complementation Test; D006801:Humans; D008956:Models, Chemical; D009154:Mutation; D010641:Phenotype; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014638:Vanadates / Q000494:pharmacology
D002352:Carrier Proteins; D027682:Cation Transport Proteins; D011993:Recombinant Fusion Proteins; D014638:Vanadates; D003300:Copper; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C081184:ATP7A protein, human; D000073840:Copper-Transporting ATPases