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

Abstract : 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-12/C-13,N-14/N-15 and H2O/D2O isotope effects in Arabidopsis GS1 catalysis and compared to the prokaryotic (Escherichia coli) enzyme. A(14)N/N-15 isotope effect (V-15/K approximate to 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 (V-15/K = 0.965), suggesting that the reaction intermediate is involved in an amidation-deamidation equilibrium favoring N-15. There was no C-12/C-13 kinetic isotope effect (V-13/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-D approximate to 0.5), showing that restructuration of the active site due to displacement of H2O by D2O facilitates the processing of intermediates. (C) 2016 Elsevier Masson SAS. All rights reserved.
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https://hal-univ-lyon1.archives-ouvertes.fr/hal-02331375
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Submitted on : Thursday, October 24, 2019 - 1:01:22 PM
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Caroline Mauve, Nicolas Giraud, Edouard R. A. Boex-Fontvieille, Ingrid Antheaume, Ilia Tea, et al.. Kinetic commitment in the catalysis of glutamine synthesis by GS1 from Arabidopsis using N-14/N-15 and solvent isotope effects. Plant Physiology and Biochemistry, 2016, 108, pp.203-211. ⟨10.1016/j.plaphy.2016.07.015⟩. ⟨hal-02331375⟩

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