RANDOM MUTAGENESIS OF GLUTAMINE-SYNTHETASE FROM ESCHERICHIA-COLI - CORRELATION BETWEEN STRUCTURE, ACTIVITY, AND FITNESS

Glutamine synthetase is a key enzyme in nitrogen metabolism and has be en improved during the course of evolution. The present level of its a ctivity was evaluated by comparing with the activities of mutant enzym es prepared by random mutagenesis. With respect to the transferase act ivity, 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 prepa rations of the wild-type enzyme (GLS-W) and two selected mutant enzyme s (GLS-H and GLS-L) showed specific transferase activities (units/mg p rotein) of 84.5, 184 and 7.1, respectively. For synthetase activity, t he k(cat) values (s(-1)) were: GLS-W, 25.8; GLS-H, 300; GLS-L, 23.2; t he 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 estim ated 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 d odecamer molecule. The distances between the active site Mn2+ ion and the alpha-carbon atoms of the mutated residues are longer than 20 Angs trom. Tyr-397 is known to be the target site for the regulation of act ivity by adenylylation. Finally, the effect of the difference in the a ctivity of glutamine synthetase on the specific growth rate of Escheri chia coli was examined. E. coli expressing GLS-W showed higher specifi c growth rate than that expressing GLS-H or GLS-L.