Biophysical studies of Escherichia coli glutamine synthetase.

The purpose of the EPR and NMR studies presented in this article was to determine the spatial relationship between the n1 and n2 metal ion sites of E. coli glutamine synthetase. Table I presents the distances between these two metal ion sites in various complexes using Mn(II) bound to each site. These studies also employed the transition-state analog, methionine sulfoximine, as an active-site probe as well as various nucleotide complexes. Two primary conclusions result from these data. The Mn(II)-Mn(II) distance changes from approximately 10 to approximately 8 A when nucleotides bind to the enzyme presumably as the result of a protein conformational change. Two Mn(II) ions can bind to the enzyme in the presence of the substitution-inert Co(III)-ATP complex, implying that the metal ion [Co(III)] coordinated to the beta-gamma phosphoryl groups in the complex is displaced from the normal n2 metal ion site. A model showing the probable spatial relationships among components of the active site is shown in Fig. 6. This model comprises our current working hypothesis of the active site of glutamine synthetase. Further studies of distance relationships are presently underway in our laboratory and will be placed in the context of this model and the known kinetic mechanism.