Biochemical parameters of glutamine synthetase from Klebsiella aerogenes

Abstract

The glutamine synthetase (GS) [EC 6.3.1.2] from K. aerogenes is similar to that from Escherichia coli in several respects: it is repressed by high levels of NH3 in the growth medium; its biosynthetic activity is greatly reduced by adenylylation; and adenylylation lowers the pH optimum and alters the response of the enzymes to various inhibitors in the .gamma.-glutamyl transferase (.gamma.GT) assay. There are several important differences: the isoactivity point for the adenylylated and non-adenylylated forms in the .gamma.GT assay occurs at pH 7.55 in K. aerogenes and at pH 7.15 in E. coli; the non-adenylylated form of the GS from K. aerogenes is stimulated by 60 mM MgCl2 in the .gamma.GT assay at pH 7.15. A biosynthetic reaction assay that correlates well with the number of non-adenylylated enzyme subunits, as determined by Mg2+ inhibition of the .gamma.GT assay, is described. Special methods must be used to harvest growing cells to prevent changes in the adenylylation state of GS from occurring during harvesting.