title: The cobalt(II)-alkaline phosphatase system at alkaline pH.
abstract: The uptake of cobalt(II) ions by apoalkaline phosphatase at pH 8 (the pH optimum for activity) has been investigated by the combined use of electronic and 1H NMR spectroscopies. The presence of fast-relaxing high spin cobalt(II) ions in the active site cavity of the protein induces sizable isotropic shifts of the 1H NMR signals of metal-coordinated protein residues, allowing us to propose a metal uptake pattern by the various metal binding sites both in the presence and in the absence of magnesium ions. In the absence of magnesium the active site is not organized in specific metal binding sites. The first equivalent of cobalt(II) ions per dimer binds in an essentially unspecific and possibly fluxional fashion, giving rise to a six-coordinated chromophore. The second and third equivalents induce the formation of increasing amounts of metal ions pairs, cooperatively arranged into the A and B sites of the same subunit with a five- and six-coordinated geometry, respectively. The fourth and fifth equivalents induce the formation of fully blocked A-B pairs in both subunits. Magnesium shows the property of organizing the metal binding sites, probably through coordination to the C sites. Electronic and 1H NMR titration with Co2+ ions show that the initial amount of fluxional cobalt is smaller than in the absence of magnesium and that A-B pairs are more readily formed. Titration of fully metalated Co4Mg2alkaline phosphatase samples with phosphate confirms binding of only one phosphate per dimer.
authors.affiliation: Department of Chemistry, University of Florence, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Banci
authors.forename: I
authors.initials: I
authors.lastname: Bertini
authors.forename: C
authors.initials: C
authors.lastname: Luchinat
authors.forename: M S
authors.initials: MS
authors.lastname: Viezzoli
authors.forename: Y J
authors.initials: YJ
authors.lastname: Wang
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism*; D001665:Binding Sites; D003035:Cobalt / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy
chemical_list: D003035:Cobalt; D000469:Alkaline Phosphatase; D008274:Magnesium