Glutamine synthetase is a key enzyme in nitrogen metabolism and has been improved during the course of evolution. The present level of its activity was evaluated by comparing with the activities of mutant enzymes prepared by random mutagenesis. With respect to the transferase activity, only 3% of a total of 7688 mutants had markedly higher activity, to the extent of 2.7 times higher than that of the wild-type. These results show that the level of the activity of the wild-type enzyme is high and well optimized, though it is not the highest. Purified preparations of the wild-type enzyme (GLS-W) and two selected mutant enzymes (GLS-H and GLS-L) showed specific transferase activities (units/mg protein) of 84.5, 184 and 7.1, respectively. For synthetase activity, the k(cat) values (s(-1)) were : GLS-W, 25.8; GLS-H, 300; GLS-L, 23.2; the K-m values (mM) for L-glutamate were : GLS-W, 2.6; GLS-H, 10.7; GLS-L, 14.6. The numbers of adenylyl groups bound to the enzyme were estimated to be : GLS-W, 11; GLS-H, 0; GLS-L, 7. GLS-H had a mutation of Tyr-397 to His, while GLS-L had two amino acid changes from Ala-35 to Val and Pro-94 to Leu. These residues are located on the surface of the dodecamer molecule. The distances between the active site Mn2+ ion and the alpha-carbon atoms of the mutated residues are longer than 20 Angstrom. Tyr-397 is known to be the target site for the regulation of activity by adenylylation. Finally, the effect of the difference in the activity of glutamine synthetase on the specific growth rate of Escherichia coli was examined. E. coli expressing GLS-W showed higher specific growth rate than that expressing GLS-H or GLS-L.