Kinetic commitment in the catalysis of glutamine synthesis by GS1 from Arabidopsis using N/N 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 C/C, N/N and HO/DO isotope effects in Arabidopsis GS1 catalysis and compared to the prokaryotic (Escherichia coli) enzyme. AN/N isotope effect (V/K ≈ 1.015, with respect to substrate NH) 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 (V/K = 0.965), suggesting that the reaction intermediate is involved in an amidation-deamidation equilibrium favoring N. There was no C/C kinetic isotope effect (V/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 (V ≈ 0.5), showing that restructuration of the active site due to displacement of HO by DO facilitates the processing of intermediates.