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Some Kinetic Properties of Bacillus subtilis Glutamine Synthetase

From the ¹ From the Laboratory of Biochemistry, Section on Enzymes, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland 20014

The glutamine synthetase of Bacillus subtilis has an absolute requirement for divalent cation. Either Mg²⁺ or Mn²⁺ will serve as an activator, but each affects various catalytic parameters differently. The concentration of either divalent cation required to produce half-maximal activity (the S_0.5) is a function of the ATP concentration. Optimal activity with Mn²⁺ occurs when the ratio of ATP to Mn²⁺ is 1.0, and decreases sharply with any deviation from this ratio. With Mg²⁺, optimal activity is achieved when the concentration of Mg²⁺ is 4- to 5-fold in excess of the ATP concentration, and it is not substantially affected by large excess of this cation. The saturation functions for the substrates are generally complex and vary with the kind of divalent cation used to activate the enzyme. With Mn²⁺, the activity increases with increasing concentration of glutamate or ammonia, but reaches a maximum at relatively low levels of substrate and then decreases with higher substrate concentrations. In contrast, with Mg²⁺, the enzyme is not saturated even at high concentration (100 mm) of either substrate. In the Mg²⁺ system, double-reciprocal plots of reaction velocity versus substrate concentration are concave downward, suggesting that negative cooperativity might be involved in substrate binding. The enzyme is inhibited by end products of glutamine metabolism, AMP, CTP, histidine, tryptophan, alanine, and glycine. Both the kinetics and sensitivity to feedback inhibition by each of these products are dependent upon whether Mg²⁺ or Mn²⁺ is used as the activating cation.

The capacity of B. subtilis glutamine synthetase to be activated by Mn²⁺ and by Mg²⁺ is apparently an intrinsic property of the enzyme; it is independent of growth conditions and is not determined by adenylylation of the enzyme as it is for the enzyme from Escherichia coli. Efforts to demonstrate adenylylated forms of the B. subtilis enzymes were unsuccessful.

In contrast to the E. coli enzyme, the glutamine synthetase from B. subtilis is inhibited by glutamine and this inhibition varies with the divalent cation used to activate the enzyme; with Mn²⁺ activation, inhibition by glutamine is greatly potentiated by AMP. From the standpoint of cellular regulation, it seems that in both E. coli and B. subtilis, the intracellular level of glutamine is the most important single factor in determining glutamine synthetase activity; however, in B. subtilis, glutamine is a direct inhibitor of glutamine synthetase activity, whereas, in E. coli, it exerts its affect indirectly through activation and inhibition of the adenylylating and deadenylylating enzymes, respectively.

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				L. V. Wray Jr. and S. H. Fisher A Feedback-resistant Mutant of Bacillus subtilis Glutamine Synthetase with Pleiotropic Defects in Nitrogen-regulated Gene Expression J. Biol. Chem., September 30, 2005; 280(39): 33298 - 33304. [Abstract] [Full Text] [PDF]