title: ADP, chloride ion, and metal ion binding to bovine brain glutamine synthetase.
abstract: The binding of divalent cations and nucleotide to bovine brain glutamine synthetase and their effects on the activity of the enzyme were investigated. In ADP-supported gamma-glutamyl transfer at pH 7.2, kinetic analyses of saturation functions gave [S]0.5 values of approximately 1 microM for Mn2+, approximately 2 mM for Mg2+, 19 nM for ADP.Mn, and 7.2 microM for ADP.Mg. The method of continuous variation applied to the Mn2+-supported reaction indicated that all subunits of the purified enzyme express activity when 1.0 equiv of ADP is bound per subunit. Measurements of equilibrium binding of Mn2+ to the enzyme in the absence and presence of ADP were consistent with each subunit binding free Mn2+ (KA approximately equal to 1.5 X 10(5) M-1) before binding the Mn.ADP complex (KA' approximately equal to 1.1 X 10(6) M-1). The binding of the first Mn2+ or Mg2+ to each subunit produces structural perturbations in the octameric enzyme, as evidenced by UV spectral and tryptophanyl residue fluorescence changes. The enzyme, therefore, has one structural site per subunit for Mn2+ or Mg2+ and a second site per subunit for the metal ion-nucleotide complex, both of which must be filled for activity expression. Chloride binding (KA' approximately equal to 10(4) M-1) to the enzyme was found to have a specific effect on the protein conformation, producing a substantial (30%) quench of tryptophanyl fluorescence and increasing the affinity of the enzyme 2-4-fold for Mg2+ or Mn2+. Arsenate, which activates the gamma-glutamyl transfer activity by binding to an allosteric site, and L-glutamate also cause conformational changes similar to those produced by Cl- binding. Anion binding to allosteric sites and divalent metal ion binding at active sites both produce tryptophanyl residue exposure and tyrosyl residue burial without changing the quaternary enzyme structure.
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002712:Chlorides / Q000378:metabolism*; D005974:Glutamate-Ammonia Ligase / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D002712:Chlorides; D008345:Manganese; D000244:Adenosine Diphosphate; D005974:Glutamate-Ammonia Ligase; D008274:Magnesium