title: Tryptophan phosphorescence as a monitor of the structural role of metal ions in alkaline phosphatase.
abstract: The phosphorescence properties of Trp109 in alkaline phosphatase from Escherichia coli have been utilized to probe the conformation of the polypeptide following the removal of metal ions, reconstitution with Zn2+ and Cd2+ and phosphorylation. The complete removal of metal ions induces a drastic loosening of the protein structure that extends to the inner core of the macromolecule. While binding of a single metal ion/subunit (A-site occupancy) restores the holoconformer, practically no structuring effect is observed upon B-site occupancy by the second incoming metal ion. An exception to this rule occurs at alkaline pH and when the adjacent subunit in the dimer is metal-free. Under these circumstances a conformation of the subunit more compact than that of the fully saturated dimer manifests some degree of communication across the subunit interface. The binding of more than two metal ions/monomer generally destabilizes the protein, the effect being more pronounced at acid pH. Finally, the binding of inorganic phosphate restores the native-like configuration abolishing any destabilization induced by excess metal ions and acid pH. If the negative cooperativity towards metal binding to A sites in doubly metalated forms at pH 8 is in substantial agreement with 113Cd-NMR data, the equivalence in conformation between Zn2+- and Cd2+-reconstituted alkaline phosphatase emphasizes that no serious structural changes are introduced by the metal replacement.
authors.affiliation: Consiglio Nazionale delle Ricerche, Istituto di Biofisica, Pisa, Italy.
authors.forename: P
authors.initials: P
authors.lastname: Cioni
authors.forename: L
authors.initials: L
authors.lastname: Piras
authors.forename: G B
authors.initials: GB
authors.lastname: Strambini
mesh_terms: D000469:Alkaline Phosphatase / Q000302:isolation & purification*; D001665:Binding Sites; D001667:Binding, Competitive; D002104:Cadmium / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology; D008163:Luminescent Measurements; D009682:Magnetic Resonance Spectroscopy; D008670:Metals / Q000032:analysis*; D010455:Peptides / Q000302:isolation & purification; D010710:Phosphates / Q000302:isolation & purification; D010766:Phosphorylation; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000032:analysis*; D015032:Zinc / Q000032:analysis
chemical_list: D008670:Metals; D010455:Peptides; D010710:Phosphates; D002104:Cadmium; D014364:Tryptophan; D000469:Alkaline Phosphatase; D015032:Zinc