Regulation and Properties of Glutamine Synthetase Purified from Bacillus cereus

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 X ATP 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 mM (Mg2+: Mn2+: ATP = 4: 0.2: 1), respectively. At each optimum pH, the Vmax values for these reactions were 6.1 (Mn2+-dependent), 7.4 (Mg2+-dependent), and 12.9 (Mg2+ plus Mn2+-dependent) mumoles per min per mg protein, respectively. Mg2+-dependent 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.

Purification and characterization of glutamine synthetase from alkalophilic Bacillus No. 170.

“…Glutamine synthetase activity was measured by the biosynthetic or r-glutamyltransferase assays as described previously (13,17). The standard biosynthetic assay mixture, in a final volume of 1.0 ml, contained 100 mM imidazole-HC1 buffer, 5 mM ATP, 100 mM L-glutamate, 10 mM ammonia, divalent cation, and sufficient enzyme.…”

“…With the pure enzyme, the protein was determined either by the method of Lowry or from the absorbance at 280 nm. Preparation of rabbit antisera for Bacillus subtilis glutamine synthetase was described previously (13). Each glutamine synthetase from B. subtilis or Escherichia coli was purified from E. coli YMC11 harboring pSGS2 (9) or pEGS1.…”

“…170. Glutamine synthetase was purified from cells grown at pH 9.0, according to the modified method previously described in Bacillus cereus enzyme (13). Cells (14 g wet weight) were suspended in four times their volume of buffer containing 50 mM Tris-HC1 (pH 7.2), 0.1 mM EDTA, and 1 mM MnC12.…”

“…In contrast, there is no evidence that the Bacillus subtilis glutamine synthetase is regulated by reversible adenylylation although it appears that the mechanisms involved are different from those in E. coli and other enterics. In earlier papers, we reported the isolation and characterization of the glutamine synthetase from both Bacillus cereus and Bacillus subtilis (12,13), the putative amino acid sequence from the DNA sequence of each gene (16,18), and the determination of the substrate binding sites by chemical modification (15,17,24) . To confirm the unique characteristic of the glutamine synthetase from these spore-forming bacteria, we have been investigating nitrogen metabolism in alkalophilic Bacillus.…”

“…In previous papers, we reported that the activity of B, subtilis and B, cereus glutamine synthetase was greatly enhanced by the addition of Mn2+ to the Mgt+-dependent assay mixture (13,17). In E. coli glutamine synthetase, the divalent cation specificity is modulated by adenylylation/deadenylylation of the subunits, and Mgt-dependent activity is inhibited by the addition of Mn2+ (21).…”