Kinetic commitment in the catalysis of glutamine synthesis by GS1 from Arabidopsis using (14)N/(15)N and solvent isotope effects.
Identifieur interne : 001C08 ( Main/Exploration ); précédent : 001C07; suivant : 001C09Kinetic commitment in the catalysis of glutamine synthesis by GS1 from Arabidopsis using (14)N/(15)N and solvent isotope effects.
Auteurs : Caroline Mauve [France] ; Nicolas Giraud [France] ; Edouard R A. Boex-Fontvieille [France] ; Ingrid Antheaume [France] ; Illa Tea [Australie] ; Guillaume Tcherkez [Australie]Source :
- Plant physiology and biochemistry : PPB [ 1873-2690 ] ; 2016.
Descripteurs français
- KwdFr :
- MESH :
- analyse : Isotopes de l'azote.
- biosynthèse : Glutamine.
- métabolisme : Arabidopsis, Glutamate-ammonia ligase, Glutamine, Protéines d'Arabidopsis.
- Cinétique, Glutamate-ammonia ligase, Isotopes du carbone, Protéines d'Arabidopsis, Solvants.
English descriptors
- KwdEn :
- Arabidopsis (metabolism), Arabidopsis Proteins (chemistry), Arabidopsis Proteins (metabolism), Carbon Isotopes (chemistry), Glutamate-Ammonia Ligase (chemistry), Glutamate-Ammonia Ligase (metabolism), Glutamine (biosynthesis), Glutamine (metabolism), Kinetics, Nitrogen Isotopes (analysis), Solvents (chemistry).
- MESH :
- chemical , analysis : Nitrogen Isotopes.
- chemical , biosynthesis : Glutamine.
- chemical , chemistry : Arabidopsis Proteins, Carbon Isotopes, Glutamate-Ammonia Ligase, Solvents.
- metabolism : Arabidopsis, Arabidopsis Proteins, Glutamate-Ammonia Ligase, Glutamine.
- Kinetics.
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 (12)C/(13)C, (14)N/(15)N and H2O/D2O isotope effects in Arabidopsis GS1 catalysis and compared to the prokaryotic (Escherichia coli) enzyme. A(14)N/(15)N isotope effect ((15)V/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 ((15)V/K = 0.965), suggesting that the reaction intermediate is involved in an amidation-deamidation equilibrium favoring (15)N. There was no (12)C/(13)C kinetic isotope effect ((13)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 ((D)V ≈ 0.5), showing that restructuration of the active site due to displacement of H2O by D2O facilitates the processing of intermediates.
DOI: 10.1016/j.plaphy.2016.07.015
PubMed: 27448794
Affiliations:
- Australie, France
- Pays de la Loire, Île-de-France
- Nantes, Orsay
- Université Paris-Sud, Université de Nantes
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UMR 5276, Université de Lyon, 69361, Lyon Cedex 07</wicri:regionArea><wicri:noRegion>69361, Lyon Cedex 07</wicri:noRegion><wicri:noRegion>69361, Lyon Cedex 07</wicri:noRegion></affiliation></author><author><name sortKey="Tea, Illa" sort="Tea, Illa" uniqKey="Tea I" first="Illa" last="Tea">Illa Tea</name><affiliation wicri:level="4"><nlm: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.</nlm:affiliation><country xml:lang="fr">Australie</country><wicri:regionArea>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</wicri:regionArea><orgName type="university">Université de Nantes</orgName><placeName><settlement type="city">Nantes</settlement><region type="region" nuts="2">Pays de la Loire</region></placeName></affiliation></author><author><name sortKey="Tcherkez, Guillaume" sort="Tcherkez, Guillaume" uniqKey="Tcherkez G" first="Guillaume" last="Tcherkez">Guillaume Tcherkez</name><affiliation wicri:level="1"><nlm: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.</nlm:affiliation><country xml:lang="fr">Australie</country><wicri:regionArea>Research School of Biology, ANU College of Medicine, Biology and Environment, Australian National University, Canberra ACT, 2601</wicri:regionArea><wicri:noRegion>2601</wicri:noRegion></affiliation></author></analytic><series><title level="j">Plant physiology and biochemistry : PPB</title><idno type="eISSN">1873-2690</idno><imprint><date when="2016" type="published">2016</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Arabidopsis (metabolism)</term><term>Arabidopsis Proteins (chemistry)</term><term>Arabidopsis Proteins (metabolism)</term><term>Carbon Isotopes (chemistry)</term><term>Glutamate-Ammonia Ligase (chemistry)</term><term>Glutamate-Ammonia Ligase (metabolism)</term><term>Glutamine (biosynthesis)</term><term>Glutamine (metabolism)</term><term>Kinetics</term><term>Nitrogen Isotopes (analysis)</term><term>Solvents (chemistry)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Arabidopsis (métabolisme)</term><term>Cinétique</term><term>Glutamate-ammonia ligase ()</term><term>Glutamate-ammonia ligase (métabolisme)</term><term>Glutamine (biosynthèse)</term><term>Glutamine (métabolisme)</term><term>Isotopes de l'azote (analyse)</term><term>Isotopes du carbone ()</term><term>Protéines d'Arabidopsis ()</term><term>Protéines d'Arabidopsis (métabolisme)</term><term>Solvants ()</term></keywords><keywords scheme="MESH" type="chemical" qualifier="analysis" xml:lang="en"><term>Nitrogen Isotopes</term></keywords><keywords scheme="MESH" type="chemical" qualifier="biosynthesis" xml:lang="en"><term>Glutamine</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Arabidopsis Proteins</term><term>Carbon Isotopes</term><term>Glutamate-Ammonia Ligase</term><term>Solvents</term></keywords><keywords scheme="MESH" qualifier="analyse" xml:lang="fr"><term>Isotopes de l'azote</term></keywords><keywords scheme="MESH" qualifier="biosynthèse" xml:lang="fr"><term>Glutamine</term></keywords><keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Arabidopsis</term><term>Arabidopsis Proteins</term><term>Glutamate-Ammonia Ligase</term><term>Glutamine</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Arabidopsis</term><term>Glutamate-ammonia ligase</term><term>Glutamine</term><term>Protéines d'Arabidopsis</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Kinetics</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Cinétique</term><term>Glutamate-ammonia ligase</term><term>Isotopes du carbone</term><term>Protéines d'Arabidopsis</term><term>Solvants</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">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 (12)C/(13)C, (14)N/(15)N and H2O/D2O isotope effects in Arabidopsis GS1 catalysis and compared to the prokaryotic (Escherichia coli) enzyme. A(14)N/(15)N isotope effect ((15)V/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 ((15)V/K = 0.965), suggesting that the reaction intermediate is involved in an amidation-deamidation equilibrium favoring (15)N. There was no (12)C/(13)C kinetic isotope effect ((13)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 ((D)V ≈ 0.5), showing that restructuration of the active site due to displacement of H2O by D2O facilitates the processing of intermediates.</div></front></TEI><affiliations><list><country><li>Australie</li><li>France</li></country><region><li>Pays de la Loire</li><li>Île-de-France</li></region><settlement><li>Nantes</li><li>Orsay</li></settlement><orgName><li>Université Paris-Sud</li><li>Université de Nantes</li></orgName></list><tree><country name="France"><region name="Île-de-France"><name sortKey="Mauve, Caroline" sort="Mauve, Caroline" uniqKey="Mauve C" first="Caroline" last="Mauve">Caroline Mauve</name></region><name sortKey="Antheaume, Ingrid" sort="Antheaume, Ingrid" uniqKey="Antheaume I" first="Ingrid" last="Antheaume">Ingrid Antheaume</name><name sortKey="Boex Fontvieille, Edouard R A" sort="Boex Fontvieille, Edouard R A" uniqKey="Boex Fontvieille E" first="Edouard R A" last="Boex-Fontvieille">Edouard R A. Boex-Fontvieille</name><name sortKey="Giraud, Nicolas" sort="Giraud, Nicolas" uniqKey="Giraud N" first="Nicolas" last="Giraud">Nicolas Giraud</name></country><country name="Australie"><region name="Pays de la Loire"><name sortKey="Tea, Illa" sort="Tea, Illa" uniqKey="Tea I" first="Illa" last="Tea">Illa Tea</name></region><name sortKey="Tcherkez, Guillaume" sort="Tcherkez, Guillaume" uniqKey="Tcherkez G" first="Guillaume" last="Tcherkez">Guillaume Tcherkez</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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