Mechanistic studies of glutamine synthetase from Escherichia coli. An integrated mechanism for biosynthesis, transferase, ATPase reaction.

The mechanism of biosynthetic, transferase, ATPase, and transphosphorylation reactions catalyzed by unadenylylated glutamine synthetase from E. coli was studied. Activation complex(es) involved in the biosynthetic reaction are produced in the presence of either Mg2+ or Mn2+ ; however, with the Mn2+-enzyme inhibition by the product, ADP, is so great that the overall forward biosynthetic reaction cannot be detected with the known assay methods. Binding studies show that substrates (except for NH3 and NH2OH which are not reported here) can bind to the enzyme in a random manner and that binding of the ATP-glutamate, ADP-Pi or ADP-arsenate pairs is strongly synergistic. Inhibition and binding studies show that the same binding site is utilized for glutamate and glutamine in biosynthetic and transferase reactions, respectively, and that a common nucleotide binding site is used for all reactions studied. Studies of the reverse biosynthetic reaction and results of fluorescent titration experiments suggest that both arsenate and orthophosphate bind at a site which overlaps the gamma-phosphate site of nucleoside triphosphate. In the reverse biosynthetic and transferase reactions, ATP serves as a substrate for the Mn2+-enzyme but not for the Mg2+-enzyme. The ATP supported transferase activity of Mn2+-enzyme is probably facilitated by the generation of ADP through ATP hydrolysis. When AMP was the only nucleotide substrate added, it was converted to ATP with concomitant formation of two equivalents of glutamate, under the reverse biosynthetic reaction conditions, and no ADP was detected. The reversibility of 180 transfer between orthophosphate and gamma-acyl group of glutamate was confirmed. ATPase activity of Mg2+ and Mn2+ unadenylylated enzymes is about the same. Both enzymes forms catalyze transphosphorylation reactions between various purine nucleoside triphosphates and nucleoside diphosphates under biosynthetic reaction conditions. The data are consistent with the hypothesis that a single active center is utilized for all reactions studied. Two stepwise mecanisms that could explain the results are discussed.