Kinetic commitment in the catalysis of glutamine synthesis by GS1 from Arabidopsis using ¹⁴N/¹⁵N and solvent isotope effects

Glutamine synthetase (GS, EC 6.3.1.2) catalyzes the production of glutamine from glutamate, ammonium and ATP. Although being essential in plants for N assimilation and recycling, kinetic commitments and transition states of the reaction have not been clearly established yet. Here, we examined ¹²C/¹³C, ¹⁴N/¹⁵N and H₂O/D₂O isotope effects in Arabidopsis GS1 catalysis and compared to the prokaryotic (Escherichia coli) enzyme. A¹⁴N/¹⁵N isotope effect (¹⁵V/K ≈ 1.015, with respect to substrate NH₄⁺) was observed in the prokaryotic enzyme, indicating that ammonium utilization (deprotonation and/or amidation) was partially rate-limiting. In the plant enzyme, the isotope effect was inverse (¹⁵V/K = 0.965), suggesting that the reaction intermediate is involved in an amidation-deamidation equilibrium favoring ¹⁵N. There was no ¹²C/¹³C kinetic isotope effect (¹³V/K = 1.000), suggesting that the amidation step of the catalytic cycle involves a transition state with minimal alteration of overall force constants at the C-5 carbon. Surprisingly, the solvent isotope effect was found to be inverse, that is, with a higher turn-over rate in heavy water (^DV ≈ 0.5), showing that restructuration of the active site due to displacement of H₂O by D₂O facilitates the processing of intermediates.