Regulation of glutamine synthetase: I. Purification and properties of glutamine synthetase from Escherichia coli

Abstract

The glutamine synthetase of Escherichia coli, which is subject to cumulative feedback inhibition by eight different products of glutamine metabolism, is also regulated by repression. The enzyme is repressed by growth on media containing readily available nitrogen, such as yeast extract-peptone or high concentrations of ammonium salts. A twentyfold derepression occurs with growth-limiting concentrations of ammonia or by growth on glutamate as the sole nitrogen source. In contrast, the level of glutamate dehydrogenase is high after growth on elevated ammonium salt levels and is low with growth on glutamate. Glutamine synthetase from derepressed cells has been purified 180-fold, and was obtained as a crystalline protein which is homogeneous in the ultracentrifuge and by disc gel electrophoresis. Apparent K m values were determined for the various substrates and activators involved in the synthesis of glutamine from ATP, glutamate, and NH 3 as well as for the enzyme-catalyzed transfer of the γ-glutamyl group from glutamine to hydroxylamine. Divalent cation activator effects were examined with respect to the pH optimum of the enzyme and utilization of substrate. Whereas Mn ++ is specifically required in the transfer reaction when high levels of hydroxylamine are used, Mg ++ will replace Mn ++ as an activator in the overall reaction resulting in glutamine formation. The ability of analogues of glutamate, ATP, ADP, and ammonia to replace the normal substrates has been explored. In contrast to glutamine synthetases from other sources, the E. coli enzyme exhibits marked specificity for the l isomer of glutamate. Analytical ultracentrifugation, amino acid analyses, manganese determination, and light-scattering measurements have been performed, and indicate that the enzyme has a molecular weight of 680,000. Glutamine synthetase may be disaggregated into 12 or 14 subunits, this occurring in the presence of 5 m guanidine, or 1 m urea and 0.01 m EDTA. Fingerprint analyses of tryptic peptides of the enzyme suggest that all of the subunits are identical.