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. M utations in the Menkes gene that affect the function of the Menkes protein may cause Menkes disease in humans, which is associated with severe systemi c copper deficiency. The catalytic mechanism of the Menkes protein, includi ng the formation of transient acylphosphate, is poorly understood. We trans fected and overexpressed wild-type and targeted mutant Menkes protein in ye ast and investigated its transient acyl phosphorylation. We demonstrated th at the Menkes protein is transiently phosphorylated by ATP in a copper-spec ific and copper-dependent manner and appears to undergo conformational chan ges 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 c opper to high affinity binding sites in the transmembrane channel, followed by ATP binding and transient phosphorylation. We propose that. putative co pper-binding sites at the N-terminal domain of the Menkes protein are impor tant as sensors of low concentrations of copper but are not essential for t he overall catalytic activity.