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Structure-function analysis of purified Enterococcus hirae CopB copper ATPase: effect of Menkes/Wilson disease mutation homologues.
Biochem J. 2001 Jul 01; 357(Pt 1):217-23.BJ

Abstract

The Enterococcus hirae CopB ATPase (EC 3.6.1.3) confers copper resistance to 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 of mutations causing Menkes and Wilson diseases, diseases of copper metabolism. Many mutations in these genes have been identified in patients. Since it has not yet been possible to purify the human copper ATPases, it has proved difficult to test the impact of mutations on ATPase function. Some mutations occur in highly conserved sequence motifs, suggesting that their effect 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 function 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. The corresponding Cys-396-->Ser CopB ATPase was unable to restore copper resistance in a CopB knock-out mutant in vivo. The purified mutant ATPase still formed an acylphosphate intermediate, but possessed no detectable ATP hydrolytic activity. The most frequent Wilson disease mutation, His-1069-->Gln, was introduced into CopB as His-480-->Gln (H480Q). This mutant CopB also failed to confer copper resistance to a CopB knock-out strain. Purified H480Q CopB formed an acylphosphate intermediate and retained a small, but significant, ATPase activity. Our results reveal that Cys-396 and His-480 of CopB are key residues for ATPase function, and similar roles are suggested for Cys-1000 and His-1069 of Menkes and Wilson ATPases respectively.

Authors+Show Affiliations

Department of Clinical Pharmacology, University of Berne, 3010 Berne, Switzerland.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

11415452

Citation

Bissig, K D., et al. "Structure-function Analysis of Purified Enterococcus Hirae CopB Copper ATPase: Effect of Menkes/Wilson Disease Mutation Homologues." The Biochemical Journal, vol. 357, no. Pt 1, 2001, pp. 217-23.
Bissig KD, Wunderli-Ye H, Duda PW, et al. Structure-function analysis of purified Enterococcus hirae CopB copper ATPase: effect of Menkes/Wilson disease mutation homologues. Biochem J. 2001;357(Pt 1):217-23.
Bissig, K. D., Wunderli-Ye, H., Duda, P. W., & Solioz, M. (2001). Structure-function analysis of purified Enterococcus hirae CopB copper ATPase: effect of Menkes/Wilson disease mutation homologues. The Biochemical Journal, 357(Pt 1), 217-23.
Bissig KD, et al. Structure-function Analysis of Purified Enterococcus Hirae CopB Copper ATPase: Effect of Menkes/Wilson Disease Mutation Homologues. Biochem J. 2001 Jul 1;357(Pt 1):217-23. PubMed PMID: 11415452.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Structure-function analysis of purified Enterococcus hirae CopB copper ATPase: effect of Menkes/Wilson disease mutation homologues. AU - Bissig,K D, AU - Wunderli-Ye,H, AU - Duda,P W, AU - Solioz,M, PY - 2001/6/21/pubmed PY - 2001/8/17/medline PY - 2001/6/21/entrez SP - 217 EP - 23 JF - The Biochemical journal JO - Biochem J VL - 357 IS - Pt 1 N2 - The Enterococcus hirae CopB ATPase (EC 3.6.1.3) confers copper resistance to 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 of mutations causing Menkes and Wilson diseases, diseases of copper metabolism. Many mutations in these genes have been identified in patients. Since it has not yet been possible to purify the human copper ATPases, it has proved difficult to test the impact of mutations on ATPase function. Some mutations occur in highly conserved sequence motifs, suggesting that their effect 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 function 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. The corresponding Cys-396-->Ser CopB ATPase was unable to restore copper resistance in a CopB knock-out mutant in vivo. The purified mutant ATPase still formed an acylphosphate intermediate, but possessed no detectable ATP hydrolytic activity. The most frequent Wilson disease mutation, His-1069-->Gln, was introduced into CopB as His-480-->Gln (H480Q). This mutant CopB also failed to confer copper resistance to a CopB knock-out strain. Purified H480Q CopB formed an acylphosphate intermediate and retained a small, but significant, ATPase activity. Our results reveal that Cys-396 and His-480 of CopB are key residues for ATPase function, and similar roles are suggested for Cys-1000 and His-1069 of Menkes and Wilson ATPases respectively. SN - 0264-6021 UR - https://www.unboundmedicine.com/medline/citation/11415452/Structure_function_analysis_of_purified_Enterococcus_hirae_CopB_copper_ATPase:_effect_of_Menkes/Wilson_disease_mutation_homologues_ L2 - https://portlandpress.com/biochemj/article-lookup/doi/10.1042/0264-6021:3570217 DB - PRIME DP - Unbound Medicine ER -