title: Role of metal-binding domains of the copper pump from Archaeoglobus fulgidus.
abstract: CopA from the extreme thermophile Archaeoglobus fulgidus is a P-type ATPase that transports Cu(+) and Ag(+) and has individual metal-binding domains (MBDs) at both N- and C-termini. We expressed and purified full-length CopA as well as constructs with MBDs deleted either individually or collectively. Cu(+) and Ag(+)-dependent ATPase assays showed that full-length CopA had submicromolar affinity for both ions, but was inhibited by concentrations above 1muM. Deletion of both MBDs had no effect on affinity but resulted in loss of this inhibition. Individual deletions implicated the N-terminal MBD in causing the inhibition at concentrations >1muM. Rates of phosphoenzyme decay indicated that neither the dephosphorylation step, nor the E1P-E2P equilibrium accounted for this inhibition, suggesting the involvement of a different catalytic step. Alternative hypotheses are discussed by which the N-terminal MBD could influence the catalytic activity of CopA.
authors.affiliation: Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY 10016, USA. rice@nysbc.org
authors.forename: William J
authors.initials: WJ
authors.lastname: Rice
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Kovalishin
authors.forename: David L
authors.initials: DL
authors.lastname: Stokes
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019611:Archaeoglobus fulgidus / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D027682:Cation Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003300:Copper / Q000378:metabolism*; D000073840:Copper-Transporting ATPases; D009841:Oligonucleotides / Q000235:genetics; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: D019843:Archaeal Proteins; D027682:Cation Transport Proteins; D009841:Oligonucleotides; D003300:Copper; D000251:Adenosine Triphosphatases; D000073840:Copper-Transporting ATPases