Activities and kinetic characteristics of glutamine synthetase from Penicillium cyclopium

The level of glutamine synthetase (GS; EC 6.3.1.2) obtained as a function of culture age of Penicillium cyclopium showed that GS activity increased continuously up to 120 hours after which the enzyme activity started to decline. Glutamine synthetase from P. cyclopium was purified to homogeneity by ammonium sulphate, diethylaminoethyl (DEAE)-cellulose and Sepharose 4B. The purified enzyme showed a single band. The pH optima for both the biosynthetic and transferase activities of the enzyme were 8.2 and 8.5, respectively. Various products such as 5'-AMP, tryptophan, alanine, glycine, and histidine inhibited both reactions of GS. However, ADP activated the transferase reaction and inhibited the biosynthetic one. The K-m values for L-glutamine were 1.4 and 5.6 mM in the biosynthetic and transferase assays, respectively. The divalent metal ion is necessary for the activity of the enzyme. Mn2+ was the most effective metal ion for transferase activity however, Mg2+ was the most effective metal ion for biosynthetic activity. Ca2+ and Mn2+ strongly inhibited Mg2+-supported biosynthetic activity. GS was quite stable in tris (hydroxymethyl) aminomethane-HCl buffer (pH 7.5) containing ethylenediaminetetraacetate (EDTA), MgCl2 and 2-mercaptoethanol (2-ME).