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Information on EC 1.4.1.13 - glutamate synthase (NADPH) and Organism(s) Escherichia coli and UniProt Accession P09832

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EC Tree
IUBMB Comments
Binds FMN, FAD, 2 [4Fe-4S] clusters and 1 [3Fe-4S] cluster. The reaction takes place in the direction of L-glutamate production. The protein is composed of two subunits, alpha and beta. The alpha subunit is composed of two domains, one hydrolysing L-glutamine to NH3 and L-glutamate (cf. EC 3.5.1.2, glutaminase), the other combining the produced NH3 with 2-oxoglutarate to produce a second molecule of L-glutamate (cf. EC 1.4.1.4, glutamate dehydrogenase [NADP+]). The beta subunit transfers electrons from the cosubstrate. The NH3 is channeled within the alpha subunit through a 31 A channel. The chanelling is very efficient and in the intact alpha-beta complex ammonia is produced only within the complex. In the absence of the beta subunit, coupling between the two domains of the alpha subunit is compromised and some ammonium can leak.
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Escherichia coli
UNIPROT: P09832
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The taxonomic range for the selected organisms is: Escherichia coli
The enzyme appears in selected viruses and cellular organisms
Reaction Schemes
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2
L-glutamate
+
NADP+
=
L-glutamine
+
2-oxoglutarate
+
NADPH
+
H+
2
+
=
+
+
+
Synonyms
glutamate synthase, gogat, nadph-dependent glutamate synthase, nadph-gogat, l-glutamate synthase, ehno1, ehno2, glutamate synthase (nadph), ph0876, ph1873, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glutamate synthetase (NADP)
-
-
-
-
glutamine amide-2-oxoglutarate aminotransferase (oxidoreductase, NADP)
-
-
-
-
glutamine-ketoglutaric aminotransferase
-
-
-
-
L-glutamate synthase
-
-
-
-
L-glutamate synthetase
-
-
-
-
L-glutamine:2-oxoglutarate aminotransferase, NADPH oxidizing
-
-
-
-
NADPH-dependent glutamate synthase
-
-
-
-
NADPH-glutamate synthase
-
-
-
-
NADPH-GOGAT
NADPH-linked glutamate synthase
-
-
-
-
synthase, glutamate (reduced nicotinamide adenine dinucleotide phosphate)
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
2 L-glutamate + NADP+ = L-glutamine + 2-oxoglutarate + NADPH + H+
show the reaction diagram
kinetic mechanism
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
redox reaction
-
-
-
-
oxidation
-
-
-
-
reduction
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
L-glutamate:NADP+ oxidoreductase (transaminating)
Binds FMN, FAD, 2 [4Fe-4S] clusters and 1 [3Fe-4S] cluster. The reaction takes place in the direction of L-glutamate production. The protein is composed of two subunits, alpha and beta. The alpha subunit is composed of two domains, one hydrolysing L-glutamine to NH3 and L-glutamate (cf. EC 3.5.1.2, glutaminase), the other combining the produced NH3 with 2-oxoglutarate to produce a second molecule of L-glutamate (cf. EC 1.4.1.4, glutamate dehydrogenase [NADP+]). The beta subunit transfers electrons from the cosubstrate. The NH3 is channeled within the alpha subunit through a 31 A channel. The chanelling is very efficient and in the intact alpha-beta complex ammonia is produced only within the complex. In the absence of the beta subunit, coupling between the two domains of the alpha subunit is compromised and some ammonium can leak.
CAS REGISTRY NUMBER
COMMENTARY hide
37213-53-9
-
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
L-glutamine + 2-oxoglutarate + NADPH + H+
L-glutamate + NADP+
show the reaction diagram
NH3 + 2-oxoglutarate + NADPH + H+
L-glutamate + NADP+
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
flavin
NADH
-
no activity with NADH
additional information
-
purified enzyme contains 30.4 mol of labile sulfide per 800,000 g of protein
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2',5'-ADP
-
act as a competitive ihibitor
2'-adenylic acid
-
competitive inhibitor of the forward reaction with NADPH as the varied substrate, uncompetitive inhibitor with alpha-ketoglutarate or L-glutamine
3-Bromo-2-oxoglutarate
-
-
4-Iodoacetamidosalicylate
ADP
-
15% inhibition at 50 mM
ATP
-
19% inhibition at 25 mM and 43% inhibition at 50 mM
BaCl2
-
61% inhibition at 50 mM
Bromopyruvate
-
-
CaCl2
-
74% inhibition at 50 mM
CdCl2
-
produces more than 60% inhibition at 5 mM
cis-aconitate
-
22% inhibition at 50 mM
CoCl2
-
produces more than 60% inhibition at 5 mM
CTP
-
9% inhibition at 25 mM and 21% inhibition at 50 mM
D-glutamate
-
50% inhibition at concentrations below 7 mM
D-methionine
-
19% inhibition at 50 mM
fumarate
-
21% inhibition at 50 mM
glycine
-
significant inhibitor
GTP
-
16% inhibition at 25 mM and 43% inhibition at 50 mM
iodoacetamide
isocitrate
-
35% inhibition at 50 mM
ITP
-
17% inhibition at 25 mM and 29% inhibition at 50 mM
KBr
-
43% inhibition at 100 mM
KCl
-
32% inhibition at 100 mM
KNO3
-
47% inhibition at 100 mM
L-2-Amino-4-oxo-5-chloropentanoic acid
-
selective inhibitor of glutamine-dependent activity
L-alanine
-
significant inhibitor
L-asparagine
-
significant inhibitor
L-aspartate
-
50% inhibition at concentrations below 7 mM
L-cysteine
-
significant inhibitor
L-glutamate
L-histidine
-
significant inhibitor
L-homoserine
-
significant inhibitor
L-malate
-
19% inhibition at 50 mM
L-methionine
-
50% inhibition at concentrations below 7 mM
L-methionine sulfone
-
competitive inhibitor vs. L-glutamine, noncompetitive inhibitor vs. alpha-ketoglutarate and uncompetitive vs. NADPH
L-serine
-
significant inhibitor
MgCl2
-
70% inhibition at 50 mM
MnCl2
-
82% inhibition at 50 mM
N-ethylmaleimide
-
-
NADP+
NADPH
-
inactivation of glutamine-dependent activity, 50% inactivation at 0.36 mM in 10 min
NiCl2
-
produces more than 60% inhibition at 5 mM
O-carbamoylserine
-
competitive inhibitor vs. L-glutamine
oxaloacetate
-
-
oxalylglycine
-
competitive inhibitor vs. alpha-ketoglutarate, noncompetitive inhibitor vs. L-glutamine and uncompetitive vs. NADPH
p-chloromercuribenzoate
-
-
p-chloromercuriphenylsulfonate
-
more than 50% loss in activity in 60 min at 0.05 mM
pyruvate
-
20% inhibition at 50 mM
succinate
-
16% inhibition at 50 mM
UTP
-
17% inhibition at 25 mM and 33% inhibition at 50 mM
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.24
2-oxoglutarate
-
NH3-dependent glutamate synthase and apoglutamate synthase
0.0047 - 0.0073
alpha-ketoglutarate
0.25
L-glutamine
-
-
0.0022 - 0.014
NADPH
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.106
2',5'-ADP
-
-
0.025
L-2-Amino-4-oxo-5-chloropentanoic acid
-
-
0.86
p-chloromercuribenzoate
-
-
additional information
additional information
-
-
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.24
-
apoenzyme lacking flavin and non-heme iron, glutamine-dependent activity
0.92
-
native enzyme, NH3-dependent activity
14.3
-
native enzyme
18.64
-
native enzyme, glutamine-dependent activity
4.45
-
apoenzyme lacking flavin and non-heme iron, NH3-dependent activity
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8.4
-
for glutamate synthesis, NH3-dependent glutamate synthase and apoenzyme
9.4
-
optimum for glutamate-dependent reduction of NADP+, NH3-dependent enzyme, apoglutamate synthase enzyme
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.4 - 9
-
at pH 6.4 and 9.0 about 50% of activity maximum
6.5 - 8.7
-
at pH 6.5 and 8.7 about 50% of activity maximum
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
SwissProt
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
135000
-
4 * 53000 + 4 * 135000, SDS-PAGE, urea polyacrylamide gel electrophoresis
53000
-
4 * 53000 + 4 * 135000, SDS-PAGE, urea polyacrylamide gel electrophoresis
800000
-
sedimentation equilibrium, gel filtration
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
octamer
-
4 * 53000 + 4 * 135000, SDS-PAGE, urea polyacrylamide gel electrophoresis
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
2-mercaptoethanol, stabilizes
-
alpha-ketoglutarate stabilizes
-
alpha-ketoglutarate, L-glutamine, L-glutamate and NADP when present together partial protect against enzyme inactivation
-
incubation with L-glutamine or L-glutamate results in marked time- and temperature-dependent loss in activity
-
NADP causes relatively small loss in activity compared with control values
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-80°C, 20 mM potassium-HEPES, pH 7.2, 2 mM alpha-ketoglutarate, 1 mM EDTA, stable for at least 6 months
-
4°C, 20 mM potassium-HEPES, pH 7.2, 2 mM alpha-ketoglutarate, 1 mM EDTA, 5 mM 2-mercaptoethanol, 20% loss of activity, after 1 month
-
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
using streptomycin sulfate treatment, ammonium sulfate fractionation, heat treatment, agarose gel filtration and DEAE-cellulose column chromatography
-
using sulfate precipitation, gel filtration and column chromatography on 2',5'-ADP-Sepharose
-
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Miller, R.E.; Stadtman, E.R.
Glutamate synthase from Escherichia coli. An iron-sulfide flavoprotein
J. Biol. Chem.
247
7407-7419
1972
Escherichia coli
Manually annotated by BRENDA team
Schmidt, C.N.G.; Jervis, L.
Affinity purification of glutamate synthase from Escherichia coli
Anal. Biochem.
104
127-129
1980
Escherichia coli
Manually annotated by BRENDA team
Rendina, A.R.; Orme-Johnson, W.H.
Glutamate synthase: on the kinetic mechanism of the enzyme from Escherichia coli W
Biochemistry
17
5388-5393
1978
Escherichia coli
Manually annotated by BRENDA team
Mntsl, P.; Zalkin, H.
Properties of apoglutamate synthase and comparison with glutamate dehydrogenase
J. Biol. Chem.
251
3300-3305
1976
Escherichia coli
Manually annotated by BRENDA team
Mntsl, P.; Zalkin, H.
Glutamate synthase. Properties of the glutamine-dependent activity
J. Biol. Chem.
251
3294-3299
1976
Escherichia coli
Manually annotated by BRENDA team
Meister, A.
Glutamate synthase from Escherichia coli, Klebsiella aerogenes, and Saccharomyces cerevisiae
Methods Enzymol.
113
327-337
1985
Klebsiella aerogenes, Escherichia coli
Manually annotated by BRENDA team
Suzuki, A.; Knaff, D.B.
Glutamate synthase: structural, mechanistic and regulatory properties, and role in the amino acid metabolism
Photosynth. Res.
83
191-217
2005
Acidithiobacillus ferrooxidans (Q56266), Acidithiobacillus ferrooxidans (Q56267), Archaeoglobus fulgidus (O29309), Azospirillum brasilense (Q05755), Azospirillum brasilense (Q05756), Azospirillum brasilense (Q59084), Bacillus subtilis (P39812), Escherichia coli (P09831), Escherichia coli (P09832), Klebsiella aerogenes, Methanocaldococcus jannaschii (Q58746), Methanococcus thermoautotrophicum (O26308), no activity in Arabidopsis thaliana, Priestia megaterium, Pseudomonas aeruginosa (P95456), Pseudomonas aeruginosa (P95457), Pyrococcus sp., Pyrococcus sp. KOD1, Rhizobium etli (Q9ZFB8), Rhizobium etli (Q9ZFB9), Rhodospirillum rubrum, Salmonella enterica subsp. enterica serovar Typhimurium (Q8ZLR3), Salmonella enterica subsp. enterica serovar Typhimurium (Q8ZLR4)
Manually annotated by BRENDA team