Structure-function analysis of purified Enterococcus hirae CopB copper ATPase: effect of Menkes/Wilson disease mutation homologues

The Enterococcus hirae CopB ATPase (EC 3.6.1.3) confers copper resistance t o the organism by expelling excess copper. Two related human ATPase genes, ATP7A (EC 3.6.1.36) and ATP7B (EC 3.6.1.36), have been cloned as the loci o f mutations causing Menkes and Wilson diseases, diseases of copper metaboli sm. Many mutations in these genes have been identified in patients. Since i t has not yet been possible to purify the human copper ATPases, it has prov ed difficult to test the impact of mutations on ATPase function. Some mutat ions occur in highly conserved sequence motifs, suggesting that their effec t on function can be tested with a homologous enzyme. Here, we used the E. hirae CopB ATPase to investigate the impact of such mutations on enzyme fun ction in vivo and in vitro. The Menkes disease mutation of Cys-1000 --> Arg , changing the conserved Cys-Pro-Cys ('CPC') motif, was mimicked in CopB. T he corresponding Cys-396 --> Ser CopB ATPase was unable to restore copper r esistance in a CopB knock-out mutant in vivo. The purified mutant ATPase st ill formed an acylphosphate intermediate, but possessed no detectable ATP h ydrolytic activity. The most frequent Wilson disease mutation, His-1069 --> Gln, was introduced into CopB as His-480 --> Gin (H480Q). This mutant CopB also failed to confer copper resistance to a CopB knock-out strain. Purifi ed H480Q CopB formed an acylphosphate intermediate and retained a small, bu t significant, ATPase activity. Our results reveal that Cys-396 and His-480 of CopB are key residues for ATPase function, and similar roles are sugges ted for Cys-1000 and His-1069 of Menkes and Wilson ATPases respectively.