Glutamine synthetase is central to nitrogen metabolism in the Gram-negative bacteria. The amount of glutamine synthetase in the cell and its catalytic activity are tightly regulated by multiple, sophisticated mechanisms. Reversible covalent modification of Tyr-397 is central to the regulation of glutamine synthetase activity, via esterification of the hydroxyl group to AMP in a process termed adenylylation. As expected, site-specific mutation of this surface-exposed Tyr-397 to Phe, Ala, or Ser was found to prevent adenylylation. Unexpectedly, these mutations had major effects on the catalytic characteristics of glutamine synthetase. The specific activities of each mutant were approximately doubled, the pH-activity profiles changed, and divalent-cation specificity was altered. Overall, Tyr397Phe behaved as if it were unadenylylated, while both Tyr397Ala and Tyr397Ser behaved as if they were adenylylated. Thus, subtle modifications in the environment of residue 397 are sufficient to induce changes previously thought to require adenylylation.