Direct evidence for separate binding sites for L-Glu and amino acid feedback inhibitors on unadenylylated glutamine synthetase from E. coli.

Glutamine synthetase from $\text{E. coli}$ is modulated by adenylylation of a tyrosine residue on each subunit of the dodecamer, as well as by feedback inhibition. With the stopped-flow fluorometric method, the binding constants for L-Glu, L-Ala, D-Val, and Gly to E_1.0—Mg, E⁷, in the absence or presence of ATP or ADP, and NH₃ were evaluated at pH 7.0, 15°. Strong synergistic effects between the amino acids and the nucleotide were observed. The fluorescence amplitude observed due to either simultaneous or sequential addition of 2 different amino acids to E or E·ATP indicate that L-Glu can bind to the enzyme simultaneously with L-Ala, Gly and D-Val; L-Ala can coexist with D-Val, Gly or D-Ala. NMR method also shows that L-Glu and L-Ala can bind simultaneously. Therefore, within our experimental conditions, the unadenylylated enzyme possesses allosteric site(s) for the amino acid inhibitors.