title: Kinetic commitment in the catalysis of glutamine synthesis by GS1 from Arabidopsis using14N/15N 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 examined12C/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 favoring15N. There was no12C/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.
authors.affiliation: Plateforme Métabolisme-Métabolome, Institute of Plant Science of Saclay (IPS2), Université Paris-Sud, 91405, Orsay Cedex, France.
authors.forename: Caroline
authors.initials: C
authors.lastname: Mauve
authors.affiliation: Institut de Chimie Moléculaire et des Matériaux d'Orsay, UMR 8182, Université Paris-Sud, 91405, Orsay Cedex, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Giraud
authors.affiliation: Laboratoire de Biotechnologies végétales appliquées aux Plantes aromatiques et médicinales, Université Jean Monnet, 42023, Saint Etienne Cedex 2, France.
authors.forename: Edouard R A
authors.initials: ERA
authors.lastname: Boex-Fontvieille
authors.affiliation: Laboratoire de Géologie de Lyon Terre, Planètes, Environnement. UMR 5276, Université de Lyon, 69361, Lyon Cedex 07, France.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Antheaume
authors.affiliation: Chimie Et Interdisciplinarité: Synthèse, Analyse, Modélisation, UMR 6230, Université de Nantes, Nantes Cedex 3, 44322, France; John Curtin School of Medical Research, ANU College of Medicine, Biology and Environment, Australian National University, Canberra ACT, 2601, Australia.
authors.forename: Illa
authors.initials: I
authors.lastname: Tea
authors.affiliation: Research School of Biology, ANU College of Medicine, Biology and Environment, Australian National University, Canberra ACT, 2601, Australia. Electronic address: guillaume.tcherkez@anu.edu.au.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Tcherkez
mesh_terms: D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000378:metabolism*; D002247:Carbon Isotopes / Q000737:chemistry; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry / Q000378:metabolism*; D005973:Glutamine / Q000096:biosynthesis* / Q000378:metabolism; D007700:Kinetics; D009587:Nitrogen Isotopes / Q000032:analysis; D012997:Solvents / Q000737:chemistry
chemical_list: D029681:Arabidopsis Proteins; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D012997:Solvents; D005973:Glutamine; C000616891:GLN1;2 protein, Arabidopsis; D005974:Glutamate-Ammonia Ligase
keywords: Amidation; Catalysis; Glutamine; Isotope effect; Kinetic commitment