Regulation and Properties of Glutamine Synthetase Purified from Bacillus cereus

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Abstract
The glutamine synthetase from Bacillus cereus IFO 3131 was purified to homogeneity. The enzyme is a dodecamer with a molecular weight of approximately 600,000, and its subunit molecular weight is 50,000. Both Mg2+ and Mn2+ activated the enzyme as to the biosynthesis of L-glutamine, but, unlike in the case of the E. coli enzyme, the Mg2+-dependent activity was stimulated by the addition of Mn2+ The highest activity was obtained when 20 mM Mg2+ and 0.5 mM Mn2+ were added to the assay mixture. For each set of optimal assay conditions, the apparent Km values for glutamate, ammonia and a divalent cation complex were 1.03, 0.34, and 0.40 mM (Mn2+ : ATP=1 : 1); 14.0, 0.47, and 0.91 mM (Mg2+ : ATP=4 : 1); and 9.09, 0.45, and 0.77 m (Mg2+ : Mn2+ : ATP=4 : 0.2 : 1), respectively. At each optimum pH, the Vm values for these reactions were 6.1 (Mn2+-dependent) 7.4 (Mg2+-dependent), and 12.9 (Mgt2+ plus Mn2+ dependent) μmoles per min per mg protein, respectively. Mg2+ glutamine synthetase activity was inhibited by the addition of AMP or glutamine; however, this inhibitory effect was suppressed in the case of the Mg2+ plus Mn2+-dependent reaction. These results suggest that the activity of the B. cereus glutamine synthetase is regulated by both the intracellular concentration and the ratio of Mn2+/Mg2+in vivo. Also in the present investigation, a potent glutamine synthetase inhibitor(s) was detected in crude extracts from B. cereus.