Bacillus subtilis glutamine synthetase. Specific catalytic changes associated with limited sulfhydryl modification.

Bacillus subtilis glutamine synthetase readily reacts with iodoacetamide, producing very specific changes in the catalytic parameters characteristic of the native enzyme. The alkylation reaction is influenced both by substrates and feedback inhibitors of the enzyme; whereas ammonia and glutamate are without effect, Mg2+-ATP enhances the alkylation reaction, and Mn2f-ATP effectively prevents the catalytic changes that accompany incubation of the enzyme with the alkylating agent. Histidine, tryptophan, and glutamine also potentiate the response of the enzyme to iodoacetamide. With limited alkylation, a marked increase in activity is seen using Mn2f as divalent cation, a sharp reduction in specific activity is seen in the assay using Mg2+ as divalent cation, and the response of the enzyme to feedback inhibitors is altered. Whereas these changes in B. subtilis catalytic activity are qualitatively similar to changes seen with enzymatic adenylylation of Escherichia coli glutamine synthetase, no role in vivo for sulfhydryl modification of B. subtilis enzyme has been found. When assayed with Mn2f as divalent cation, alkylated glutamine synthetase has a higher apparent K, for glutamate than does native enzyme; the substrate saturation function with the alkylated enzyme is hyperbolic and has lost the inhibition by high concentrations of glutamate, which is characteristic of the native enzyme. When assayed with MgZf as divalent cation, the alkylated enzyme shows loss of apparent aiTinity for glutamate at high glutamate concentrations. Sulfhydryl titration indicates that 1 cysteine residue is readily alkylated per subunit, with two to three additional groups titrated after dissociation of the enzyme. The alkylation reaction is specific for cysteine residues. These results thus provide by chemical modification of the B. subtilis glutamine synthetase a system that is qualitatively analogous to the enzymatic adenylylation of the E. coli glutamine synthetase; the results also exhibit a clear structural distinction between the two enzymes. Under similar conditions of study, the sulfhydryl groups of the B. subtilis enzyme are readily titrated by iodoacetamide, whereas those of E. coli glutamine synthetase are not accessible to the alkylating agent.