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Information on EC 1.4.1.3 - glutamate dehydrogenase [NAD(P)+] and Organism(s) Homo sapiens
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1.4.1.3 glutamate dehydrogenase [NAD(P)+]Specify your search results
The taxonomic range for the selected organisms is: Homo sapiens
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria, Archaea
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria, Archaea
Synonyms
hgdh2, glutamate dehydrogenase 1, gdhii, nad(p)-dependent glutamate dehydrogenase, nad(p)+-dependent glutamate dehydrogenase, legdh1, nad(p)-glutamate dehydrogenase, nad(p)h-dependent glutamate dehydrogenase, ttgdh, 
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topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
dehydrogenase, glutamate (nicotinamide adenine dinucleotide (phosphate))
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GDH
GDH1
GDH2
GLUD1
GLUD2
glutamate dehydrogenase
glutamate dehydrogenase 1
glutamate dehydrogenase 2
glutamic acid dehydrogenase
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glutamic dehydrogenase
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hGDH1
hGDH2
L-glutamate dehydrogenase
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L-glutamic acid dehydrogenase
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Legdh1
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Membrane protein 50
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MP50
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NAD(P)-glutamate dehydrogenase
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NAD(P)H-dependent glutamate dehydrogenase
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NAD(P)H-utilizing glutamate dehydrogenase
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GDH
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
oxidation
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redox reaction
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reduction
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reductive amination
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PATHWAY SOURCE
PATHWAYS
KEGG
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-, -, -, -
SYSTEMATIC NAME
IUBMB Comments
L-glutamate:NAD(P)+ oxidoreductase (deaminating)
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CAS REGISTRY NUMBER
COMMENTARY
9029-12-3
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
2-oxoglutarate + NADH + NH3
L-glutamate + NAD+ + H2O
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-
-
-
?
L-glutamate + H2O + NAD(P)+
2-oxoglutarate + NH3 + NAD(P)H + H+
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-
-
-
?
L-glutamate + H2O + NAD(P)+
2-oxoglutarate + NH3 + NAD(P)H + H+
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-
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r
L-glutamate + H2O + NAD(P)+
2-oxoglutarate + NH3 + NAD(P)H + H+
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-
-
r
additional information
?
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in vitro, the thermodynamic equilibrium of mammalian GDH favors glutamate synthesis. Because the GDH-catalyzed reaction is reversible, its direction is expected to depend on the concentration of the substrates and the affinity of the enzyme (Km value) for these substrates. In addition to substrate concentrations, the GDH catalysis is affected by the pH, the ionic strength and the composition of the buffer
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-
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additional information
?
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in vitro, the thermodynamic equilibrium of mammalian GDH favors glutamate synthesis. Because the GDH-catalyzed reaction is reversible, its direction is expected to depend on the concentration of the substrates and the affinity of the enzyme (Km value) for these substrates. In addition to substrate concentrations, the GDH catalysis is affected by the pH, the ionic strength and the composition of the buffer
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additional information
?
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in vitro, the thermodynamic equilibrium of mammalian GDH favors glutamate synthesis. Because the GDH-catalyzed reaction is reversible, its direction is expected to depend on the concentration of the substrates and the affinity of the enzyme (Km value) for these substrates. In addition to substrate concentrations, the GDH catalysis is affected by the pH, the ionic strength and the composition of the buffer
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
L-glutamate + H2O + NAD(P)+
2-oxoglutarate + NH3 + NAD(P)H + H+
-
-
-
r
L-glutamate + H2O + NAD(P)+
2-oxoglutarate + NH3 + NAD(P)H + H+
-
-
-
r
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
NADH
-
-
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
AlCl3
-
increase in sensitivity to aluminium as pH decreases, inhibitory effect is predominant below pH 7.0, no effect above pH 8.5. Completely inactivated enzyme contains 2 mol of aluminum per mol of subunit. Citrate, NaF, N-(2-hydroxyethyl) ethylenediaminetriacetic acid or EDTA efficiently protects against inactivation. Citrate and NaF release aluminum from the completely inactivated aluminum-enzyme complex and fully recover enzyme activity. Binding of aluminum induces a decrease in alpha helices and beta sheets and an increase in random coil
ATP
-
wild-type: inhibition at 0.1 mM and between 0.5-1.0 mM and above, activation at 1 mM, H454Y and S448P mutant enzyme: activation between 0.1-1 mM, inhibition above, R463A mutant enzyme: progressive inhibition between 0.01 and 10 mM
Chloroquine
-
potent inhibitor of isozymes GDH1 and GDH2 at a dose-dependent manner, the inhibitory effect of chloroquine on GDH2 is abolished by the presence of ADP and L-leucine, whereas GTP does not change the sensitivity to chloroquine inhibition, shows a non-competitive inhibition against 2-oxoglutarate and an uncompetitive inhibition against NADH
NAD+
-
incubation with 0.1 mM for 60 min inhibits hGDH1 and hGDH2 by 75% and 70%, respectively, incubations for longer time periods up to 3 h, does not further increase the inhibition of hGDH isoenzymes, ADP-ribosylated hDGH isozymes are reactivated by Mg2+-dependent mitochondrial ADP-ribosylcysteine hydrolase
p-chloromercuribenzoic acid
-
progressive decrease in enzyme activity of both isoenzymes, inhibition is not affected by addition of GTP or ADP
GTP
inhibition of wild-type enzyme and mutant enzymes r470H and N498S. No inhibition of mutant enzyme G456A. IC50 of wild-type enzyme is 0.00019 mM, IC50 of mutant enzyme G456A is 0.0028 mM, IC50 of mutant enzyme R470G is 0.00017 mM, IC50 of mutant enzyme N498S is 0.0002 mM
GTP
-
IC50 for wild-type GLUD1: 0.0122 mM, IC50 for mutant enzyme R443S: 0.0162 mM, IC50 for mutant enzyme M415L: 0.0147 mM, IC50 for mutant enzyme M370L: 0.0113 mM, IC50 for mutant enzyme S331T: 0.0108 mM
GTP
potent inhibitor. IC50 for wild-type enzyme is 0.00019 mM, IC50 for mutant enzyme G456A is 0.0028 mM. ADP renders the GLUD1-derived enzyme less sensitive to GTP inhibition; totally insensitive to, it becomes amenable to GTP inhibition in presence of ADP
GTP
-
efective inhibitor: activity below 10% of its full activity; GDH2 is resistent to GTP inhibition
palmitoyl-CoA
-
concentration-dependent inhibition, GDH1 is less sensitive to palmitoyl-CoA inhibition than GDH2
additional information
the isozyme hGDH2 is resistant against GTP inhibition
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additional information
the isozyme hGDH2 is resistant against GTP inhibition
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additional information
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the isozyme hGDH2 is resistant against GTP inhibition
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
ATP
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wild-type: activation at 1 mM, inhibition below and above, H454Y and S448P mutant enzymes: progressive increase in activity until 10 mM, inhibition above
ADP
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1 mM, pH 8.0, 3fold activation of wild-type enzyme, no significant actication of Tyr187 mutant enzymes
ADP
in absence of GTP the isoenzyme GLUD2 assumes a conformational state associated with little catalytic activity, it remains amenable to full activation by ADP and/or L-Leu
ADP
ADP is an activator of hGDH1 and hGDH2 which binds only to the open state
ADP
ADP is an activator of hGDH1 and hGDH2 which binds only to the open state. hGDH1mutant R443S/G456A exhibits a reduced ADP sensitivity compared both to hGDH1 and to hGDH2. The addition of the M415L and R470H mutations results in an even more pronounced drop in ADP affinity
L-Leu
0.3-10 mM stimulates isoenzyme GLUD2 to a greater extent than isoenzyme GLUD1
L-Leu
0.3-10 mM stimulates isoenzyme GLUD2 to a greater extent than isoenzyme GLUD1. In absence of GTP the isoenzyme GLUD2 assumes a conformational state associated with little catalytic activity, it remains amenable to full activation by ADP and/or L-Leu
leucine
-
the enzyme is subject to allosteric activation by leucine invlving ARg134, and Asp185, structural mechanism, overview
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1.25
2-oxoglutarate
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isozyme GDH1, in the presence of 1 mM ADP, in 50 mM triethanolamine, pH 8.0, 100 mM ammonium acetate, 0.1 mM NADH, and 2 mM EDTA, pH 8.0, at 25°C
1.25
2-oxoglutarate
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wild type isozyme GDH1, 50 mM triethanolamine pH 8.0, 100 mM ammonium acetate, 0.1 mM NADH, and 2 mM EDTA, pH 8.0 at 25°C
1.27
2-oxoglutarate
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mutant enzyme L415M/S443R/A456G, 50 mM triethanolamine pH 8.0, 100 mM ammonium acetate, 0.1 mM NADH, and 2 mM EDTA, pH 8.0 at 25°C
1.29
2-oxoglutarate
-
mutant enzyme M415L/R443S/G456A, 50 mM triethanolamine pH 8.0, 100 mM ammonium acetate, 0.1 mM NADH, and 2 mM EDTA, pH 8.0 at 25°C
1.39
2-oxoglutarate
-
isozyme GDH1, in the presence of 1 mM ADP, in 50 mM triethanolamine, pH 8.0, 100 mM ammonium acetate, 0.1 mM NADH, and 2 mM EDTA, pH 8.0, at 25°C
1.39
2-oxoglutarate
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wild type isozyme GDH2, 50 mM triethanolamine pH 8.0, 100 mM ammonium acetate, 0.1 mM NADH, and 2 mM EDTA, pH 8.0 at 25°C
1.5
2-oxoglutarate
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mutant enzyme R443S/G456A, in 50 mM triethanolamine buffer (pH 8.0), 2.6 mM EDTA, 1.4 mM NADP+, and 1 mM ADP
2
2-oxoglutarate
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wild type isozyme GDH1, in 50 mM triethanolamine buffer (pH 8.0), 2.6 mM EDTA, 1.4 mM NADP+, and 1 mM ADP
2.1
2-oxoglutarate
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wild type isozyme GDH2, in 50 mM triethanolamine buffer (pH 8.0), 2.6 mM EDTA, 1.4 mM NADP+, and 1 mM ADP
10.7
L-glutamate
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wild type isozyme GDH2, in 50 mM triethanolamine buffer (pH 8.0), 2.6 mM EDTA, 1.4 mM NADP+, and 1 mM ADP
10.8
L-glutamate
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mutant enzyme R443S/G456A, in 50 mM triethanolamine buffer (pH 8.0), 2.6 mM EDTA, 1.4 mM NADP+, and 1 mM ADP
12.4
L-glutamate
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wild type isozyme GDH1, in 50 mM triethanolamine buffer (pH 8.0), 2.6 mM EDTA, 1.4 mM NADP+, and 1 mM ADP
0.075
NADH
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mutant enzyme L415M/S443R/A456G, 50 mM triethanolamine pH 8.0, 100 mM ammonium acetate, 0.1 mM NADH, and 2 mM EDTA, pH 8.0 at 25°C
0.08
NADH
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mutant enzyme M415L/R443S/G456A, 50 mM triethanolamine pH 8.0, 100 mM ammonium acetate, 0.1 mM NADH, and 2 mM EDTA, pH 8.0 at 25°C
0.081
NADH
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isozyme GDH1, in the presence of 1 mM ADP, in 50 mM triethanolamine, pH 8.0, 100 mM ammonium acetate, 0.1 mM NADH, and 2 mM EDTA, pH 8.0, at 25°C
0.081
NADH
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wild type isozyme GDH1, 50 mM triethanolamine pH 8.0, 100 mM ammonium acetate, 0.1 mM NADH, and 2 mM EDTA, pH 8.0 at 25°C
0.086
NADH
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isozyme GDH2, in the presence of 1 mM ADP, in 50 mM triethanolamine, pH 8.0, 100 mM ammonium acetate, 0.1 mM NADH, and 2 mM EDTA, pH 8.0, at 25°C
0.086
NADH
-
wild type isozyme GDH2, 50 mM triethanolamine pH 8.0, 100 mM ammonium acetate, 0.1 mM NADH, and 2 mM EDTA, pH 8.0 at 25°C
13.4
NH3
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wild type isozyme GDH1, in 50 mM triethanolamine buffer (pH 8.0), 2.6 mM EDTA, 1.4 mM NADP+, and 1 mM ADP
17.1
NH3
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wild type isozyme GDH2, in 50 mM triethanolamine buffer (pH 8.0), 2.6 mM EDTA, 1.4 mM NADP+, and 1 mM ADP
22.2
NH3
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mutant enzyme R443S/G456A, in 50 mM triethanolamine buffer (pH 8.0), 2.6 mM EDTA, 1.4 mM NADP+, and 1 mM ADP
additional information
additional information
-
Km-values for 2-oxoglutaratefor mutant enzyme R443S, recombinant wild-type enzyme and wild-type enzyme from human liver
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additional information
additional information
-
lowering the pH of the buffer from pH 8.0 to pH 7.0 increases the Km for ammonia substantially, i.e. for hGDH1 from 12.8 mM to 57.5 mM, and for hGDH2: from 14.7 mM to 62.2 mM, thus essentially precluding reductive amination
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additional information
additional information
when assayed in triethanolamine buffer, pH 8.0, at 1.0 mM ADP, hGDH1 and hGDH2 show similar catalytic properties (Vmax and Kms for 2-oxoglutarate, ammonia and glutamate), kinetics, overview
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additional information
additional information
when assayed in triethanolamine buffer, pH 8.0, at 1.0 mM ADP, hGDH1 and hGDH2 show similar catalytic properties (Vmax and Kms for 2-oxoglutarate, ammonia and glutamate), kinetics, overview
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additional information
additional information
-
when assayed in triethanolamine buffer, pH 8.0, at 1.0 mM ADP, hGDH1 and hGDH2 show similar catalytic properties (Vmax and Kms for 2-oxoglutarate, ammonia and glutamate), kinetics, overview
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
101
NADH
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mutant enzyme M415L/R443S/G456A, 50 mM triethanolamine pH 8.0, 100 mM ammonium acetate, 0.1 mM NADH, and 2 mM EDTA, pH 8.0 at 25°C
104
NADH
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isozyme GDH1, in the presence of 1 mM ADP, in 50 mM triethanolamine, pH 8.0, 100 mM ammonium acetate, 0.1 mM NADH, and 2 mM EDTA, pH 8.0, at 25°C
104
NADH
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wild type isozyme GDH1, 50 mM triethanolamine pH 8.0, 100 mM ammonium acetate, 0.1 mM NADH, and 2 mM EDTA, pH 8.0 at 25°C
118
NADH
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mutant enzyme L415M/S443R/A456G, 50 mM triethanolamine pH 8.0, 100 mM ammonium acetate, 0.1 mM NADH, and 2 mM EDTA, pH 8.0 at 25°C
130
NADH
-
isozyme GDH2, in the presence of 1 mM ADP, in 50 mM triethanolamine, pH 8.0, 100 mM ammonium acetate, 0.1 mM NADH, and 2 mM EDTA, pH 8.0, at 25°C
130
NADH
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wild type isozyme GDH2, 50 mM triethanolamine pH 8.0, 100 mM ammonium acetate, 0.1 mM NADH, and 2 mM EDTA, pH 8.0 at 25°C
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0015
17beta-estradiol
Homo sapiens
pH 8.0, temperature not specified in the publication
0.0151
17beta-estradiol
Homo sapiens
presence of 0.1 M ADP, pH 8.0, temperature not specified in the publication
0.0269
17beta-estradiol
Homo sapiens
pH 8.0, temperature not specified in the publication
0.0692
17beta-estradiol
Homo sapiens
presence of 0.1 M ADP, pH 8.0, temperature not specified in the publication
0.001
corticosterone
Homo sapiens
pH 8.0, temperature not specified in the publication
0.0021
corticosterone
Homo sapiens
pH 8.0, temperature not specified in the publication
0.0025
corticosterone
Homo sapiens
presence of 0.1 M ADP, pH 8.0, temperature not specified in the publication
0.0032
corticosterone
Homo sapiens
presence of 0.1 M ADP, pH 8.0, temperature not specified in the publication
0.0072
dehydroepiandrosterone
Homo sapiens
pH 8.0, temperature not specified in the publication
0.0294
dehydroepiandrosterone
Homo sapiens
presence of 0.1 M ADP, pH 8.0, temperature not specified in the publication
0.216
dehydroepiandrosterone
Homo sapiens
pH 8.0, temperature not specified in the publication
0.396
dehydroepiandrosterone
Homo sapiens
presence of 0.1 M ADP, pH 8.0, temperature not specified in the publication
0.0494
dehydrotestosterone
Homo sapiens
pH 8.0, temperature not specified in the publication
0.0915
dehydrotestosterone
Homo sapiens
presence of 0.1 M ADP, pH 8.0, temperature not specified in the publication
0.494
dehydrotestosterone
Homo sapiens
pH 8.0, temperature not specified in the publication
0.814
dehydrotestosterone
Homo sapiens
presence of 0.1 M ADP, pH 8.0, temperature not specified in the publication
0.001
diethylstilbestrol
Homo sapiens
presence of 0.1 M ADP, pH 8.0, temperature not specified in the publication
0.00167
diethylstilbestrol
Homo sapiens
pH 8.0, temperature not specified in the publication
0.0071
diethylstilbestrol
Homo sapiens
presence of 0.1 M ADP, pH 8.0, temperature not specified in the publication
0.008
diethylstilbestrol
Homo sapiens
pH 8.0, temperature not specified in the publication
0.0113
estriol
Homo sapiens
pH 8.0, temperature not specified in the publication
0.145
estriol
Homo sapiens
pH 8.0, temperature not specified in the publication
0.189
estriol
Homo sapiens
presence of 0.1 M ADP, pH 8.0, temperature not specified in the publication
0.316
estriol
Homo sapiens
presence of 0.1 M ADP, pH 8.0, temperature not specified in the publication
0.00017
GTP
Homo sapiens
inhibition of wild-type enzyme and mutant enzymes r470H and N498S. No inhibition of mutant enzyme G456A. IC50 of mutant enzyme R470G is 0.00017 mM, IC50 of mutant enzyme N49
0.00019
GTP
Homo sapiens
potent inhibitor. IC50 for wild-type enzyme is 0.00019 mM
0.00019
GTP
Homo sapiens
inhibition of wild-type enzyme and mutant enzymes r470H and N498S. No inhibition of mutant enzyme G456A. IC50 of wild-type enzyme is 0.00019 mM
0.0028
GTP
Homo sapiens
IC50 for mutant enzyme G456A is 0.0028 mM. ADP renders the GLUD1-derived enzyme less sensitive to GTP inhibition
18.5
GTP
Homo sapiens
-
GDH1, in the presence of 1 mM ADP, in 50 mM triethanolamine pH 8.0 buffer
78.5
GTP
Homo sapiens
-
GDH2, in the absence of ADP, in 50 mM triethanolamine pH 8.0 buffer in 50 mM TRA pH 8.0 buffer
139.4
GTP
Homo sapiens
-
mutant enzyme R443S/G456A, in the absence of ADP, in 50 mM triethanolamine pH 8.0 buffer
166.8
GTP
Homo sapiens
-
GDH2, in the presence of 1 mM ADP, in 50 mM triethanolamine pH 8.0 buffer
622.4
GTP
Homo sapiens
-
mutant enzyme R443S/G456A, in the absence of ADP, in 50 mM triethanolamine pH 8.0 buffer
0.0117
pregnenolone
Homo sapiens
pH 8.0, temperature not specified in the publication
0.0541
pregnenolone
Homo sapiens
presence of 0.1 M ADP, pH 8.0, temperature not specified in the publication
0.104
pregnenolone
Homo sapiens
pH 8.0, temperature not specified in the publication
0.287
pregnenolone
Homo sapiens
presence of 0.1 M ADP, pH 8.0, temperature not specified in the publication
0.0123
progesterone
Homo sapiens
pH 8.0, temperature not specified in the publication
0.0589
progesterone
Homo sapiens
presence of 0.1 M ADP, pH 8.0, temperature not specified in the publication
0.119
progesterone
Homo sapiens
pH 8.0, temperature not specified in the publication
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
100
-
recombinant R463A mutant enzyme, maximal activity in the presence of 6 mM leucine
130
140
-
recombinant H454Y mutant enzyme, maximal activity in the presence of 0.2 mM ADP
55
-
recombinant S448P mutant enzyme, maximal activity in the presence of 6 mM leucine
69
-
recombinant S448P mutant enzyme, maximal activity in the presence of 0.2 mM ADP
additional information
130
-
recombinant wild-type enzyme, maximal activity in the presence of 0.2 mM ADP or 6 mM leucine
130
-
recombinant S448P mutant enzyme, maximal activity in the presence of 6 mM leucine
additional information
-
hGDH1 displays a high basal activity of 40% of its maximal activity. Regulation of the hGDH1 activity is achieved by interplay of GTP and ADP/L-leucine
additional information
-
hGDH2 displays a basal activity less than 10% of its maximal activity. Control of the hGDH2 activity depends on the level of its basal activity and on changing ADP/L-leucine concentrations. For hGDH2 enzyme protein concentration and basal activity show a linear pattern of dependence. Lowering the temperature of the reaction mixture from 25°C to 20°C has a stabilizing effect on hGDH2, as under these conditions, the specific basal activities of the enzyme increases. Increasing the concentration of the TrisHCl buffer from 50 mM to 125 mM or to 250 mM (pH 8.0) raises basal activity levels, but the maximal activity declines
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.5
7.8
7.5
-
in TRA buffer hGDH2 displays its maximal activity and its highest basal activity at pH 7.5
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7 - 8
7 - 8
-
lowering the pH of the buffer from pH 8.0 to pH 7.0 increases the Km for ammonia substantially, i.e. for hGDH1 from 12.8 mM to 57.5 mM, and for hGDH2: from 14.7 mM to 62.2 mM, thus essentially precluding reductive amination
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
gene expression profiling reveals that GLUD2 is selectively and consistently upregulated in glioma cells harboring IDH mutations
both hGDH1 and hGDH2 are densely expressed in estrogen producing cells
both hGDH1 and hGDH2 are densely expressed in estrogen producing cells
additional information
-
the enzyme is immunologically distinct from those of other mammalian brains
upregulation of hGDH1/2 expression occurs in cancer
additional information
GDH2 tissue distribution, immunohistochemic analysis using a specific antibody generated with the 12-amino acid hGDH2-specific peptide PTAEFQDSISGA, corresponding to residues 436-447 of the mature human protein, overview
additional information
-
GDH2 tissue distribution, immunohistochemic analysis using a specific antibody generated with the 12-amino acid hGDH2-specific peptide PTAEFQDSISGA, corresponding to residues 436-447 of the mature human protein, overview
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
-
almost one third of GLDH serum originates from rough endoplasmic reticulum
-
more than two third of GLDH serum originates from mitochondria
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
drug target
the ADP-ribosylation of glutamate dehydrogenase is catalyzed by Sirt4, and downregulates the TCA cycle. In the ternary complex model of Sirt4-NAD+-GDH, the acetylated lysine 171 of GDH is located close to NAD+. This suggests a possible mechanism underlying the ADP-ribosylation at cysteine 172, which may occur through a transient intermediate with ADP-ribosylation at the acetylated lysine 171
evolution
malfunction
metabolism
physiological function
additional information
evolution
-
while GDH in most mammals is encoded by a single GLUD1 gene, humans and other primates have acquired a GLUD2 gene with distinct tissue expression profile
evolution
hGDH2 emerged recently via retroposition during primate evolution, being only present in humans and some closely related great apes. Functional evolution of hGDH isoenzymes, overview. Reflecting the very recent emergence of hGDH2 from hGDH1, the two human proteins show very high amino acid sequence homology (about 97%), differing in only 15 of 505 amino acids in their mature forms. Despite this similarity, hGDH2 has unique enzymatic and regulatory properties. These include GTP resistance and low basal activity amenable to activation by ADP and/or L-leucine, lower optimal pH and relative sensitivity to thermal inactivation. These properties are to a large extent associated with only two of the 15 amino acid substitutions that occurred in the course of hGDH2 evolution. In particular, the Gly456 to Ala substitution confers GTP resistance, whereas the Arg443 to Ser change is associated with lower basal activity, though still permitting activation by ADP
evolution
reflecting the very recent emergence of hGDH2 from hGDH1, the two human proteins show very high amino acid sequence homology (about 97%), differing in only 15 of 505 amino acids in their mature forms. Despite this similarity, hGDH2 has unique enzymatic and regulatory properties
evolution
while most mammals possess a single GDH1 protein (hGDH1 in the human) that is highly expressed in the liver, humans and other primates have acquired, via duplication, an hGDH2 isoenzyme with distinct functional properties and tissue expression profile. hGDH2 underwent rapid evolutionary adaptation, acquiring unique properties that enable enhanced enzyme function under conditions inhibitory to its ancestor hGDH1. These are thought to provide a biological advantage to humans with hGDH2 evolution occurring concomitantly with human brain development
evolution
while most mammals possess a single GDH1 protein (hGDH1 in the human) that is highly expressed in the liver, humans and other primates have acquired, via duplication, an hGDH2 isoenzyme with distinct functional properties and tissue expression profile. hGDH2 underwent rapid evolutionary adaptation, acquiring unique properties that enable enhanced enzyme function under conditions inhibitory to its ancestor hGDH1. These are thought to provide a biological advantage to humans with hGDH2 evolution occurring concomitantly with human brain development. A major evolutionary adaptation of hGDH2 is the ability of the enzyme to downregulate its activity in the absence of allosteric effectors
malfunction
deregulation of hGDH1/2 is implicated in the pathogenesis of several human disorders
malfunction
deregulation of hGDH1/2 is implicated in the pathogenesis of several human disorders. Glioma cells with the R132H IDH1 mutation show selective inhibition of GLUD2 expression markedly slows cell growth. xpression of GLUD2 (but not GLUD1) promotes tumor expansion, suggesting that R132H IDH1 glioma cells proliferate by utilizing enhanced glutamate flux through the GLUD2 pathway
metabolism
glutamate dehydrogenase pathway and its roles in cell and tissue biology in health and disease, , glutamate dehydrogenase (GDH) pathway and the Krebs cycle function, oxidative deamination of glutamate by hGDH1 and hGDH2 generates 2-oxoglutarate, ammonia and NADH orNADPH, regulation of the isozymes, detailed overview
metabolism
glutamate dehydrogenase pathway and its roles in cell and tissue biology in health and disease, glutamate dehydrogenase (GDH) pathway and the Krebs cycle function, oxidative deamination of glutamate by hGDH1 and hGDH2 generates 2-oxoglutarate, ammonia and NADH orNADPH, regulation of the isozymes, detailed overview
physiological function
-
influence of alcohol on leukocyte GLDH activity, its diagnostic value, influence on metabolism and cells toxicity is analysed. Examination is conducted in 238 alcoholics and in 244 healthy persons. A fast increase of leukocyte GLDH activity after break in alcohol consumption is found. After 24 hours, activity increases by 21.8% (median 31.6%), after seven days by 33% (median 52%), yet after a short interval since last alcohol intake (up to 48 hours), it increases by 32% (median 36%)
physiological function
the glutamate dehydrogenase catalyzes the reversible interconversion of glutamate to 2-oxoglutarate and ammonia using NADP(H) and NAD(H) as cofactors, thus interconnecting amino acid and carbohydrate metabolism. Mammalian GDH is allosterically regulated, with GTP and ADP being the main negative and positive modulators, respectively
physiological function
isozyme hGDH2 is found in both human astrocytes and neurons, where it is thought to contribute to glutamate handling, both as a neurotransmitter and as a metabolic intermediate. It plays a putative role in early nervous system development, neurodegenerative processes, and oncogenesis. Regarding its role in cancer pathophysiology, hGDH2 promotes tumor cell survival especially under deprived conditions, such as glucose or glutamine depletion
physiological function
the enzyme catalyzes the reversible conversion of glutamate to 2-oxoglutarate and ammonia while reducing NAD(P)+ to NAD(P)H serving both catabolic and anabolic reactions. In mammalian tissues, oxidative deamination of glutamate via GDH generates 2-oxoglutarate, which is metabolized by the Krebs cycle, leading to the synthesis of ATP. In addition, the GDH pathway is linked to diverse cellular processes, including ammonia metabolism, acid-base equilibrium, redox homeostasis (via formation of fumarate), lipid biosynthesis (via oxidative generation of citrate), and lactate production. hGDH2 is co-expressed with hGDH1 in human brain, kidney, testis and steroidogenic organs, but not in the liver. In human cerebral cortex, hGDH1 and hGDH2 are expressed in astrocytes, the cells responsible for removing and metabolizing transmitter glutamate, and for supplying neurons with glutamine and lactate. In human testis, hGDH2 (but not hGDH1) is densely expressed in the Sertoli cells, known to provide the spermatids with lactate and other nutrients. In steroid producing cells, hGDH1/2 is thought to generate reducing equivalents (NADPH) in the mitochondria for the biosynthesis of steroidal hormones. Lastly, up-regulation of hGDH1/2 expression occurs in cancer, permitting neoplastic cells to utilize glutamine/glutamate for their growth. In addition to contributing to Krebs cycle anaplerosis and energy production, GDH function is linked to redox homeostasis and cell signaling processes. By regulating bioenergetics and redox homeostasis human GDH1/2 have emerged as key players in the pathogenesis of human neoplasias and as therapeutic targets for halting tumor development and expansion
physiological function
the enzyme catalyzes the reversible conversion of glutamate to 2-oxoglutarate and ammonia while reducing NAD(P)+ to NAD(P)H serving both catabolic and anabolic reactions. In mammalian tissues, oxidative deamination of glutamate via GDH generates 2-oxoglutarate, which is metabolized by the Krebs cycle, leading to the synthesis of ATP. In addition, the GDH pathway is linked to diverse cellular processes, including ammonia metabolism, acid-base equilibrium, redox homeostasis (via formation of fumarate), lipid biosynthesis (via oxidative generation of citrate), and lactate production. hGDH2 is co-expressed with hGDH1 in human brain, kidney, testis and steroidogenic organs, but not in the liver. In human cerebral cortex, hGDH1 and hGDH2 are expressed in astrocytes, the cells responsible for removing and metabolizing transmitter glutamate, and for supplying neurons with glutamine and lactate. In human testis, hGDH2 (but not hGDH1) is densely expressed in the Sertoli cells, known to provide the spermatids with lactate and other nutrients. In steroid producing cells, hGDH1/2 is thought to generate reducing equivalents (NADPH) in the mitochondria for the biosynthesis of steroidal hormones. Lastly, upregulation of hGDH1/2 expression occurs in cancer, permitting neoplastic cells to utilize glutamine/glutamate for their growth. In addition to contributing to Krebs cycle anaplerosis and energy production, GDH function is linked to redox homeostasis and cell signaling processes. By regulating bioenergetics and redox homeostasis human GDH1/2 have emerged as key players in the pathogenesis of human neoplasias and as therapeutic targets for halting tumor development and expansion
additional information
GDH1 has an advanced structure that also encompasses the antenna showing that the entire hexamer undergoes substantial conformational changes during each catalytic cycle. As the catalytic cleft opens the NAD+ domain moves away from the glutamate binding domain, twisting around the antenna in a clockwise direction along with concomitant clockwise rotation of the ascending alpha-helix of the antenna. In addition, the small alpha-helix of the antenna (at the end of its descending random coil) undergoes striking conformational changes as the catalytic mouth opens. The importance of this small helix is underscored by observations showing that mutation of amino acids located in this helix in hGDH1 attenuate GTP inhibition leading to hyperinsulinemia/hyperammonemia (HI/HA) syndrome
additional information
GDH1 has an advanced structure that also encompasses the antenna showing that the entire hexamer undergoes substantial conformational changes during each catalytic cycle. As the catalytic cleft opens the NAD+ domain moves away from the glutamate binding domain, twisting around the antenna in a clockwise direction along with concomitant clockwise rotation of the ascending alpha-helix of the antenna. In addition, the small alpha-helix of the antenna (at the end of its descending random coil) undergoes striking conformational changes as the catalytic mouth opens. The importance of this small helix is underscored by observations showing that mutation of amino acids located in this helix in hGDH1 attenuate GTP inhibition leading to hyperinsulinemia/hyperammonemia (HI/HA) syndrome
additional information
-
GDH1 has an advanced structure that also encompasses the antenna showing that the entire hexamer undergoes substantial conformational changes during each catalytic cycle. As the catalytic cleft opens the NAD+ domain moves away from the glutamate binding domain, twisting around the antenna in a clockwise direction along with concomitant clockwise rotation of the ascending alpha-helix of the antenna. In addition, the small alpha-helix of the antenna (at the end of its descending random coil) undergoes striking conformational changes as the catalytic mouth opens. The importance of this small helix is underscored by observations showing that mutation of amino acids located in this helix in hGDH1 attenuate GTP inhibition leading to hyperinsulinemia/hyperammonemia (HI/HA) syndrome
additional information
structure-function analysis, overview. Structure comparison with isozyme hGDH1, comparison of open, semi-closed and closed conformations from apo-hGDH1 (PDB ID 1L1F) and hGDH2 (PDB ID 6G2U)
additional information
structure-function analysis, overview. Structure comparison with isozyme hGDH1, comparison of open, semi-closed and closed conformations from apo-hGDH1 (PDB ID 1L1F) and hGDH2 (PDB ID 6G2U)
additional information
-
structure-function analysis, overview. Structure comparison with isozyme hGDH1, comparison of open, semi-closed and closed conformations from apo-hGDH1 (PDB ID 1L1F) and hGDH2 (PDB ID 6G2U)
additional information
structure-function analysis, overview. Structure comparison with isozyme hGDH2, comparison of open, semi-closed and closed conformations from apo-hGDH1 (PDB ID 1L1F) and hGDH2 (PDB ID 6G2U)
additional information
structure-function analysis, overview. Structure comparison with isozyme hGDH2, comparison of open, semi-closed and closed conformations from apo-hGDH1 (PDB ID 1L1F) and hGDH2 (PDB ID 6G2U)
additional information
-
structure-function analysis, overview. Structure comparison with isozyme hGDH2, comparison of open, semi-closed and closed conformations from apo-hGDH1 (PDB ID 1L1F) and hGDH2 (PDB ID 6G2U)
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). DHE3_HUMAN
558
0
61398
Swiss-Prot
Mitochondrion (Reliability: 5)
DHE4_HUMAN
558
0
61434
Swiss-Prot
Mitochondrion (Reliability: 1)
E9KL48_HUMAN
558
0
61398
TrEMBL
Mitochondrion (Reliability: 5)
Q9UQV0_HUMAN
39
0
4441
TrEMBL
other Location (Reliability: 4)
Q14400_HUMAN
258
0
28695
TrEMBL
other Location (Reliability: 4)
A0A140VK14_HUMAN
558
0
61434
TrEMBL
Mitochondrion (Reliability: 1)
Q59FQ4_HUMAN
299
0
32323
TrEMBL
other Location (Reliability: 1)
Q9BSD0_HUMAN
264
0
29290
TrEMBL
other Location (Reliability: 5)
Q53GW3_HUMAN
558
0
61340
TrEMBL
Mitochondrion (Reliability: 5)
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
330000
-
wild-type enzyme and mutant enzymes K333L, K337L, K344L, K346L, S445L and G446L, gel filtration
56000
56500
56000
-
6 * 56000, wild-type enzyme and mutant enzymes K333L, K337L, K344L, K346L, S445L and G446L
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
-
the GdhA-GdhB-Leu complex is crystallized as a heterohexamer composed of four GdhA subunits and two GdhB subunits. In this complex, six leucine molecules are bound at subunit interfaces identified as glutamate-binding sites in the GdhB-Glu complex
hexamer
homohexamer
additional information
hexamer
-
6 * 56000, wild-type enzyme and mutant enzymes K333L, K337L, K344L, K346L, S445L and G446L
hexamer
-
the GdhB-Glu complex is a hexamer that binds 12 glutamate molecules: six molecules are bound at the substrate-binding sites, and the remaining six are bound at subunit interfaces, each composed of three subunits
homohexamer
dimer of trimers, the enzyme adopts a novel semi-closed conformation, which is an intermediate between known open and closed GDH1 conformations, differing from both. Structure-function analysis, overview. Each monomer consists of the glutamate-binding domain (residues 1-210), the NAD+-binding domain (residues 211-399), the antenna (residues 400-448), the pivot helix (residues 449-478) and the C-terminal alpha-helix (residues 479-501), which are very similar to the structural features of hGDH1. The antenna region is formed by the ascending and descending helix
additional information
-
Thermus thermophilus, possesses GDH with a unique subunit configuration composed of two different subunits, GdhA, the regulatory subunit, and GdhB, the catalytic subunit, structure of the GdhA-GdhB-Leu complex, overview
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
the ADP-ribosylation of glutamate dehydrogenase is catalyzed by Sirt4, and downregulates the TCA cycle. In the ternary complex model of Sirt4-NAD+-GDH, the acetylated lysine 171 of GDH is located close to NAD+. This suggests a possible mechanism underlying the ADP-ribosylation at cysteine 172, which may occur through a transient intermediate with ADP-ribosylation at the acetylated lysine 171
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purified isozyme hGDH2, hGDH2 is crystallized in the absence of allosteric regulators or active site ligands, by hanging drop vapour diffusion method, mixing of 0.002 ml of 9.7 mg/ml protein solution with an equal volume of reservoir solution containing 8% PEG 8000, 15% MPD, 0.4 M NaCl and 0.1 M phosphate, pH 7.0, and equilibration against 1.0 ml of reservoir solution at 18°C, method screening and optimization, X-ray diffraction structure determination and analysis at 2.9 A resolution, molecular replacement method and modelling using the hexameric human apo-enzyme hGDH1 structure (PDB ID 1L1F) as a search model
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
A456G
-
mutant of isoenzyme hGDH2 shows no change in heat inactivation process compared to wild-type enzyme
D172Y
D185A
-
site-directed mutagenesis, the mutant shows no activation by leucine in contrast to the wild-type enzyme
E279G
G446C
-
a one-month-old boy with a rare form of congenital hyperinsulinism characterised by hypoglycaemia and hyperammonaemia is described. The patient is heterozygous for a novel de novo mutation in the GLUD1 gene in exon 11 of chromosome 10, which encodes glutamate dehydrogenase (GDH). This point mutation alters the corresponding guanine-guanine-thymine (GGT) codon to thymine-guaninethymine (TGT), changing the glycine at position 446 to cysteine (Gly446Cys), which is located on the allosteric domain of the enzyme. The result confirmed the diagnosis of hyperinsulinism and hyperammonaemia syndrome. The patient is treated with diazoxide (12 mg/kg/day) and the glucose infusion is gradually decreased over four days. Blood glucose is maintained around 4 mmol/l. However, the infants ammonia level remain above 120 mmol/l
G446D
-
kinetic parameters are almost identical to that of the wild-type enzyme. Subunit composition and polymerisation process are not affected by matagenesis
G456A
G96Y
H454Y
H470R
-
mutant of isoenzyme hGDH2 shows no change in heat inactivation process compared to wild-type enzyme
K118Y
K130Y
K333L
-
kinetic parameters are almost identical to that of the wild-type enzyme. Subunit composition and polymerisation process are not affected by matagenesis
K337L
-
kinetic parameters are almost identical to that of the wild-type enzyme. Subunit composition and polymerisation process are not affected by matagenesis
K344L
-
kinetic parameters are almost identical to that of the wild-type enzyme. Subunit composition and polymerisation process are not affected by matagenesis
K346L
-
kinetic parameters are almost identical to that of the wild-type enzyme. Subunit composition and polymerisation process are not affected by matagenesis
K450E
-
mutation in the pivot helix, mutant shows diminished basal activity and a strongly decreased maximal activity, no activation by L-leucine, ADP restores the decreased activity of K450E but this occurs at substantially higher concentrations compared to wild-type, mutant shows an increased resistance to GTP inhibition, mutation makes the enzyme extremely heat-labile compared to wild-type. IC50 (GTP): 180
K450G
-
mutant enzyme is unable to bind GTP, no difference in sensitivity to aluminum binding between wild-type and mutant enzyme
K94Y
L415M
-
mutant of isoenzyme hGDH2 shows no change in heat inactivation process compared to wild-type enzyme
L415M/S443R/A456G
-
triple mutant hGDH2(hGDH1390-465)hGDH2 (amino acid segment 390-465 of hGDH2 replaced by the corresponding hGDH1 segment)
M370L
-
mutation does not abolish basal activity and does not abrogate the activation of the enzyme by L-Leu
M415L
-
mutation does not abolish basal activity and does not abrogate the activation of the enzyme by L-Leu
M415L/R443S/G456A
-
triple mutant hGDH1(hGDH2390-465)hGDH1 (amino acid segment 390-465 of hGDH1 replaced by the corresponding hGDH2 segment)
Q441R
R151M
-
site-directed mutagenesis, the mutant shows reduced activation by leucine compared to the wild-type enzyme
R443S
R470H
S331T
-
mutation does not abolish basal activity and does not abrogate the activation of the enzyme by L-Leu
S443R
-
mutant of isoenzyme hGDH2 shows a dramatic increase in thermal stability from 45 min at 45°C for the wild-type enzyme to 300 min for the mutant enzyme. KM-values and turnover-numbers are nearly identical to wild-type enzyme
S445A
site-directed mutagenesis, the specific antibody, generated from 12-amino acid hGDH2-specific peptide PTAEFQDSISGA, corresponding to residues 436-447 of the mature human protein, shows reduced reactivity with the enzyme mutant
S445L
S448P
Y187E
-
KM-values for NADH and 2-oxoglutareta are similar to wild-type values, about 4fold decrease of Vmax
Y187G
Y187M
-
KM-values for NADH and 2-oxoglutareta are similar to wild-type values, about 4fold decrease of Vmax, no significant actication by ADP
Y187S
-
KM-values for NADH and 2-oxoglutareta are similar to wild-type values, about 4fold decrease of Vmax, no significant actication by ADP
Y197R
-
KM-values for NADH and 2-oxoglutareta are similar to wild-type values, about 4fold decrease of Vmax, no significant actication by ADP
additional information
D172Y
-
ratio of turnover number to Km-value for NAD+ is 1.22fold lower than the wild-type value. Ratio of turnover number to Km-value for glutamate is 1.2fold lower than the wild-type value, isoenzyme hGDH1
D172Y
-
ratio of turnover number to Km-value for NAD+ is 1.2fold lower than the wild-type value. Ratio of turnover number to Km-value for glutamate is 1.1fold lower than the wild-type value, reduced sensitivity to ADP activation, isoenzyme hGDH2
E279G
-
mutant enzyme is unable to bind NAD+, no difference in sensitivity to aluminum binding between wild-type and mutant enzyme
G456A
mutation renders the enzyme markedly resistant to GTP inhibition, mutation abolishes the cooperative behavior of the enzyme
G456A
naturally occuring mutation, an evolutionary amino acid substitution compared to hGDH1 which confers GTP resistance, residue 456 from the pivot helix has a key role in the transition between closed and open conformations, a process which includes movements of the pivot helix and the NAD+-binding domain, leading to the opening of the active site cleft. Local flexibility is affected by an intersubunit hydrophobic interaction at the base of the antenna between residues Phe387 and Leu401. In GDH1, flexibility is also affected by the presence of the small and flexible Gly456 residue which packs against the Phe and Leu. Replacement of Gly by the bulkier and less flexible Ala456 in hGDH2 is expected to reduce local flexibility, and thus to affect the opening and closing of the active site cleft
G456A
site-directed mutagenesis, residue 456 from the pivot helix has a key role in the transition between closed and open conformations, a process which includes movements of the pivot helix and the NAD+-binding domain, leading to the opening of the active site cleft. Local flexibility is affected by an intersubunit hydrophobic interaction at the base of the antenna between residues Phe387 and Leu401. In GDH1, flexibility is also affected by the presence of the small and flexible Gly456 residue which packs against the Phe and Leu. Replacement of Gly by the bulkier and less flexible Ala456 as in hGDH2 is expected to reduce local flexibility, and thus to affect the opening and closing of the active site cleft
G96Y
-
ratio of turnover number to Km-value for NAD+ is 1.4fold lower than the wild-type value. Ratio of turnover number to Km-value for glutamate is 13.5fold lower than the wild-type value, isoenzyme hGDH1
G96Y
-
ratio of turnover number to Km-value for NAD+ is 1.4fold lower than the wild-type value. Ratio of turnover number to Km-value for glutamate is 14.3fold lower than the wild-type value, isoenzyme hGDH2
H454Y
-
mutation results in depolymerization of hexameric enzyme into active trimers. Mutation has no effect on expression or stability of the protein. The Km-value for NADH is 1.5fold greater than the wild-type value and the KM-value for 2-oxoglutarate is 2.5fold greater than the wild-type value. Vmax values are similar for wild-type and mutant enzyme
H454Y
-
mutation in the pivot helix, mutant shows diminished basal activity and a strongly decreased maximal activity, almost no activation by L-leucine, mutant H454Y requires higher concentrations of ADP for its activation than the wild-type, mutant shows an increased resistance to GTP inhibition, mutation makes the enzyme extremely heat-labile compared to wild-type. IC50 (GTP): 2.92
K118Y
-
ratio of turnover number to Km-value for NAD+ is 1.8fold lower than the wild-type value. Ratio of turnover number to Km-value for glutamate is 9.9fold lower than the wild-type value, isoenzyme hGDH1
K118Y
-
ratio of turnover number to Km-value for NAD+ is 2fold lower than the wild-type value. Ratio of turnover number to Km-value for glutamate is 8.1fold lower than the wild-type value, isoenzyme hGDH2
K130Y
-
ratio of turnover number to Km-value for NAD+ is 26.5fold lower than the wild-type value. Ratio of turnover number to Km-value for glutamate is 35fold lower than the wild-type value, isoenzyme hGDH2
K130Y
-
ratio of turnover number to Km-value for NAD+ is 41.6fold lower than the wild-type value. Ratio of turnover number to Km-value for glutamate is 47.3fold lower than the wild-type value, isoenzyme hGDH1
K94Y
-
ratio of turnover number to Km-value for NAD+ is 5.6fold lower than the wild-type value. Ratio of turnover number to Km-value for glutamate is 25.5fold lower than the wild-type value, isoenzyme hGDH2
K94Y
-
ratio of turnover number to Km-value for NAD+ is 6.6fold lower than the wild-type value. Ratio of turnover number to Km-value for glutamate is 37.8fold lower than the wild-type value, isoenzyme hGDH1
Q441R
-
mutation in the small helix of the antenna, basal activity increased by 2fold compared to wild-type, potentiated activation by L-leucine, Q411R substitution has little effect on the allosteric regulation of the mutant by ADP and GTP compared to wild-type, mutation makes the enzyme more resistant to thermal inactivation compared to wild-type. IC50 (GTP): 0.227
Q441R
site-directed mutagenesis, the specific antibody, generated from 12-amino acid hGDH2-specific peptide PTAEFQDSISGA, corresponding to residues 436-447 of the mature human protein, shows reduced reactivity with the enzyme mutant
R443S
mutation abolishes basal activity and renders the enzyme dependent on ADP for function
R443S
-
mutation abolishes basal activity and totally abrogates the activation of the enzyme by L-Leu, 1-10 mM, in absence of other effectors. When ADP, 0.025-0.1 mM, is present, L-Leu (0.3-6.0 mM) activates the mutant enzyme up to 2000%. The mutant enzyme is much less sensitive to ADP than the wild-type enzyme, however at 1 mM ADP the Vmax is comparable with that of wild-type enzyme GLUD1 GDH. KM-value for 2-oxoglutarate is similar to wild-type value
R443S
naturally occuring mutation, an evolutionary amino acid substitution which is associated with lower basal activity, though still permitting activation by ADP. Replacement of the GDH1 residue Arg443 by Ser in GDH2 was a key event early in the evolution of the GLUD2 gene in humans and great apes, which has been related with a lower basal activity and heat sensitivity. Arg443 is involved in the stabilization of open and closed conformations of GDH1. GDH1 structures reveal that the transition from open to closed conformations is associated with partial unfolding of the C-terminus of the descending helix, immediately after residue Arg443, thereby reducing the length of the helix by almost a half turn. Arg443 forms conserved intersubunit hydrogen bonds with backbone and/or side chains of Asp408 and Ser409 residues from the ascending helix of a neighbouring chain and a conserved stacking interaction with Tyr405. Through these interactions, Arg443 establishes crucial intersubunit connections that mutually stabilize the ascending and descending helices. In contrast, substitution of Arg443 by Ser in hGDH2 leads to a loss of the above interactions, and a loose packing in the antenna region. This results in a higher flexibility in the area and in a lower stability of the enzyme, which subsequently may contribute to a lower enzymatic basal activity and an increased heat sensitivity
R443S
site-directed mutagenesis, replacement of the GDH1 residue Arg443 by Ser in GDH2 was a key event early in the evolution of the GLUD2 gene in humans and great apes, which has been related with a lower basal activity and heat sensitivity. Arg443 is involved in the stabilization of open and closed conformations of GDH1. GDH1 structures reveal that the transition from open to closed conformations is associated with partial unfolding of the C-terminus of the descending helix, immediately after residue Arg443, thereby reducing the length of the helix by almost a half turn. Arg443 forms conserved intersubunit hydrogen bonds with backbone and/or side chains of Asp408 and Ser409 residues from the ascending helix of a neighbouring chain and a conserved stacking interaction with Tyr405. Through these interactions, Arg443 establishes crucial intersubunit connections that mutually stabilize the ascending and descending helices. In contrast, substitution of Arg443 by Ser in hGDH2 leads to a loss of the above interactions, and a loose packing in the antenna region. This results in a higher flexibility in the area and in a lower stability of the enzyme, as we observed in earlier studies, which subsequently may contribute to a lower enzymatic basal activity and an increased heat sensitivity
R470H
naturally occuring mutation, an evolutionary amino acid substitution
S445L
-
kinetic parameters are almost identical to that of the wild-type enzyme. Subunit composition and polymerisation process are not affected by matagenesis
S445L
-
mutation in the small helix of the antenna, basal activity increased by 2fold compared to wild-type, potentiated activation by L-leucine, S445L mutant retains the regulatory properties of the wild-type concerning its activation by ADP and inhibition by GTP, mutation makes the enzyme more resistant to thermal inactivation compared to wild-type. IC50 (GTP): 0.317
S448P
-
unstable in Tris-buffer especially in the absence of allosteric activators, basal and maximal specific activity is lower than that from wild-type
S448P
-
mutation located in the junction of the antenna with the pivot helix, mutant shows reduced basal activity without significantly altering the allosteric regulation by GTP or ADP, mutant is slightly induced by L-leucine. IC50 (GTP): 0.186
Y187G
-
KM-values for NADH and 2-oxoglutareta are similar to wild-type values, about 4fold decrease of Vmax, no significant actication by ADP
Y187G
-
mutant enzyme is unable to bind ADP, no difference in sensitivity to aluminum binding between wild-type and mutant enzyme
additional information
-
the catalytic properties of the chimeric enzymes GDH1(GDH2390-448)GDH1 and GDH2(GDH1390-448)GDH2 are not altered compared to the wild type enzyme and show almost identical sensitivity to palmitoyl-CoA inhibitory aspects of the original wild type isozymes
additional information
neither FLAG nor (His)6 tags disturb the mitochondrial localization of GDH. The addition of the small tags to the N-terminus of the mature mitochondrial enzyme does not change the ADP activation or GTP inhibition pattern of the proteins
additional information
neither FLAG nor (His)6 tags disturb the mitochondrial localization of GDH. The addition of the small tags to the N-terminus of the mature mitochondrial enzyme does not change the ADP activation or GTP inhibition pattern of the proteins
additional information
-
neither FLAG nor (His)6 tags disturb the mitochondrial localization of GDH. The addition of the small tags to the N-terminus of the mature mitochondrial enzyme does not change the ADP activation or GTP inhibition pattern of the proteins
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
45
47.5
-
GDH2 has a half-life of 38 min at 47.5°C, GDH1 has a half-life of 348 min at 47.5°C, high concentrations of phosphate (300 mM) have a protective effect on the thermal denaturation of both wild type hGDHs, this effect being more pronounced for GDH2
additional information
45
-
half-life of hGDH2 is 45 min in absence of allosteric regulators, hGDH2 is stable for 100 min in presence of 3 mM L-Leu and 1 mM ADP, mutant enzyme hGDH2 S443R has a half life of 300 min
45
-
the half life of isozyme GDH1 is 310 min at 45°C, the half life of isozyme GDH2 is 45 min at 45°C
additional information
-
hGDH1: much slower heat inactivation processes in presence of 1 mM ADP or 3 mM L-Leu
additional information
-
much slower heat inactivation processes in presence of 1 mM ADP or 3 mM L-Leu
additional information
-
Q441R or S445L mutation makes the enzyme more resistant to thermal inactivation compared to wild-type, K450E or H454Y mutation makes the enzyme extremely heat-labile compared to wild-type
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate fractionation, phenyl-Sepharose column chromatography, and hydroxyapatite chromatography
-
recombinant isozyme hGDH1 mutants from Spodoptera frugiperda SF21 cells by ammonium sulfate fractionation, hydrophobic interaction chromatography, and hydroxyapatite chromatography
recombinant isozyme hGDH2 from Spodoptera frugiperda SF21 cells by ammonium sulfate fractionation, hydrophobic interaction chromatography, and hydroxyapatite chromatography
recombinant wild-type GLUD1 and GLUD2 from Spodoptera frugiperda Sf21 cells by ammonim sulfate fractionation, and hydrophobic interaction and hydroxyapatite chromatography
-
using ammonium sulphate fractionation, hydrophobic interaction (using a phenylsepharose high performance column) and hydroxyapatite chromatography
-
wild-type and mutant enzyme, Q-Sepharose, omega-aminopentyl column, GTP-agarose
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
coexpression of wild-type and mutant enzymes with GroES and GroEl in Escherichia coli
-
gene GLUD1, recombinant expression of mutant enzymes in Spodoptera frugiperda SF21 cells
gene GLUD2, expression of 12-amino acid GDH2-specific peptide PTAEFQDSISGA, corresponding to residues 436-447 of the mature human protein, in Spodoptere frugiperda Sf21 cells using the baculovirus transfection method
gene GLUD2, recombinant expression of wild-type enzyme in Spodoptera frugiperda SF21 cells
recombinant expression of wild-type GLUD1 and GLUD2 in Spodoptera frugiperda Sf21 cells using the baculovirus expression system
-
wild-type and mutant enzyme, expression in Sf21 cells using a baculovirus expression system
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
medicine
additional information
-
C323 plays an important role in catalysis by human GDH isozymes
medicine
-
serum GLDH activity is an ideal marker of alcoholism since it is elevated in alcohol abuse but its activity declines promptly after the last alcohol intake
medicine
by regulating bioenergetics and redox homeostasis human GDH1/2 have emerged as key players in the pathogenesis of human neoplasias and as therapeutic targets for halting tumor development and expansion
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Smith, E.L.; Austen, B.M.; Blumenthal, K.M.; Nyc, J.F.
Glutamate dehydrogenase
The Enzymes, 3rd Ed. (Boyer, P. D. , ed. )
11
293-367
1975
Acholeplasma laidlawii, Auxenochlorella pyrenoidosa, Bos taurus, Gallus gallus, Homo sapiens, Lithobates catesbeianus, Rattus norvegicus, Squalus acanthias, Sus scrofa, Thunnus thynnus
-
Fang, J.; Hsu, B.Y.L.; MacMullen, C.M.; Poncz, M.; Smith, T.J.; Stanley, C.A.
Expression, purification and characterization of human glutamate dehydrogenase (GDH) allosteric regulatory mutations
Biochem. J.
363
81-87
2002
Homo sapiens
Yoon, H.Y.; Lee, E.Y.; Cho, S.W.
Cassette mutagenesis and photoaffinity labeling of adenine binding domain of ADP regulatory site within human glutamate dehydrogenase
Biochemistry
41
6817-6823
2002
Homo sapiens
Yoon, H.Y.; Cho, E.H.; Yang, S.J.; Lee, H.J.; Huh, J.W.; Choi, M.M.; Cho, S.W.
Reactive amino acid residues involved in glutamate-binding of human glutamate dehydrogenase isozymes
Biochimie
86
261-267
2004
Homo sapiens
Yang, S.J.; Huh, J.W.; Lee, J.E.; Choi, S.Y.; Kim, T.U.; Cho, S.W.
Inactivation of human glutamate dehydrogenase by aluminum
Cell. Mol. Life Sci.
60
2538-2546
2003
Homo sapiens
Jang, S.H.; Kim, A.Y.; Bahn, J.H.; Eum, W.S.; Kim, D.W.; Park, J.; Lee, K.S.; Kang, T.C.; Won, M.H.; Kang, J.H.; Kwon, O.S.; Yoon, H.Y.; Lee, E.Y.; Cho, S.W.; Choi, S.Y.
Human glutamate dehydrogenase is immunologically distinct from other mammalian orthologues
Exp. Mol. Med.
35
249-256
2003
Homo sapiens
Lee, E.Y.; Huh, J.W.; Yang, S.J.; Choi, S.Y.; Cho, S.W.; Choi, H.J.
Histidine 454 plays an important role in polymerization of human glutamate dehydrogenase
FEBS Lett.
540
163-166
2003
Homo sapiens
Yang, S.J.; Huh, J.W.; Hong, H.N.; Kim, T.U.; Cho, S.W.
Important role of Ser443 in different thermal stability of human glutamate dehydrogenase isozymes
FEBS Lett.
562
59-64
2004
Homo sapiens
Zaganas, I.; Plaitakis, A.
Single amino acid substitution (G456A) in the vicinity of the GTP binding domain of human housekeeping glutamate dehydrogenase markedly attenuates GTP inhibition and abolishes the cooperative behavior of the enzyme
J. Biol. Chem.
277
26422-26428
2002
Homo sapiens (P00367), Homo sapiens
Yoon, H.Y.; Cho, E.H.; Kwon, H.Y.; Choi, S.Y.; Cho, S.W.
Importance of glutamate 279 for the coenzyme binding of human glutamate dehydrogenase
J. Biol. Chem.
277
41448-41454
2002
Homo sapiens
Zaganas, I.; Spanaki, C.; Karpusas, M.; Plaitakis, A.
Substitution of Ser for Arg-443 in the regulatory domain of human housekeeping (GLUD1) glutamate dehydrogenase virtually abolishes basal activity and markedly alters the activation of the enzyme by ADP and L-leucine
J. Biol. Chem.
277
46552-46558
2002
Homo sapiens
Smith, T.J.; Schmidt, T.; Fang, J.; Wu, J.; Siuzdak, G.; Stanley, C.A.
The structure of apo human glutamate dehydrogenase details subunit communication and allostery
J. Mol. Biol.
318
765-777
2002
Homo sapiens (P00367)
Plaitakis, A.; Spanaki, C.; Mastorodemos, V.; Zaganas, I.
Study of structure-function relationships in human glutamate dehydrogenases reveals novel molecular mechanisms for the regulation of the nerve tissue-specific (GLUD2) isoenzyme
Neurochem. Int.
43
401-410
2003
Homo sapiens (P00367), Homo sapiens (P49448)
Choi, M.M.; Huh, J.W.; Yang, S.J.; Cho, E.H.; Choi, S.Y.; Cho, S.W.
Identification of ADP-ribosylation site in human glutamate dehydrogenase isozymes
FEBS Lett.
579
4125-4130
2005
Homo sapiens
Yang, S.J.; Cho, E.H.; Choi, M.M.; Lee, H.J.; Huh, J.W.; Choi, S.Y.; Cho, S.W.
Critical role of the cysteine 323 residue in the catalytic activity of human glutamate dehydrogenase isozymes
Mol. Cell
19
97-103
2005
Homo sapiens
Kravos, M.; Malesic, I.
Kinetics and isoforms of serum glutamate dehydrogenase in alcoholics
Alcohol Alcohol.
43
281-286
2008
Homo sapiens
Choi, M.M.; Hwang, E.Y.; Kim, E.A.; Huh, J.W.; Cho, S.W.
Identification of amino acid residues responsible for different GTP preferences of human glutamate dehydrogenase isozymes
Biochem. Biophys. Res. Commun.
368
742-747
2008
Homo sapiens
Choi, M.M.; Kim, E.A.; Choi, S.Y.; Kim, T.U.; Cho, S.W.; Yang, S.J.
Inhibitory properties of nerve-specific human glutamate dehydrogenase isozyme by chloroquine
J. Biochem. Mol. Biol.
40
1077-1082
2007
Homo sapiens
Choi, M.M.; Kim, E.A.; Yang, S.J.; Choi, S.Y.; Cho, S.W.; Huh, J.W.
Amino acid changes within antenna helix are responsible for different regulatory preferences of human glutamate dehydrogenase isozymes
J. Biol. Chem.
282
19510-19517
2007
Homo sapiens
Kanavouras, K.; Mastorodemos, V.; Borompokas, N.; Spanaki, C.; Plaitakis, A.
Properties and molecular evolution of human GLUD2 (neural and testicular tissue-specific) glutamate dehydrogenase
J. Neurosci. Res.
85
1101-1109
2007
Homo sapiens
Bahi-Buisson, N.; El Sabbagh, S.; Soufflet, C.; Escande, F.; Boddaert, N.; Valayannopoulos, V.; Bellane-Chantelot, C.; Lascelles, K.; Dulac, O.; Plouin, P.; de Lonlay, P.
Myoclonic absence epilepsy with photosensitivity and a gain of function mutation in glutamate dehydrogenase
Seizure
17
658-664
2008
Homo sapiens
Kravos, M.; Malesic, I.
Changes in leukocyte Glutamate Dehydrogenase activity in alcoholics upon break in alcohol consumption
Clin. Biochem.
43
272-277
2009
Homo sapiens
Kanavouras, K.; Borompokas, N.; Latsoudis, H.; Stagourakis, A.; Zaganas, I.; Plaitakis, A.
Mutations in human GLUD2 glutamate dehydrogenase affecting basal activity and regulation
J. Neurochem.
109 Suppl 1
167-173
2009
Homo sapiens
Chik, K.K.; Chan, C.W.; Lam, C.W.; Ng, K.L.
Hyperinsulinism and hyperammonaemia syndrome due to a novel missense mutation in the allosteric domain of the glutamate dehydrogenase 1 gene
J. Paediatr. Child Health
44
517-519
2008
Homo sapiens
Zaganas, I.; Kanavouras, K.; Mastorodemos, V.; Latsoudis, H.; Spanaki, C.; Plaitakis, A.
The human GLUD2 glutamate dehydrogenase: localization and functional aspects
Neurochem. Int.
55
52-63
2009
Homo sapiens
Spanaki, C.; Zaganas, I.; Kleopa, K.A.; Plaitakis, A.
Human GLUD2 glutamate dehydrogenase is expressed in neural and testicular supporting cells
J. Biol. Chem.
285
16748-16756
2010
Homo sapiens (P49448), Homo sapiens
Tomita, T.; Kuzuyama, T.; Nishiyama, M.
Structural basis for leucine-induced allosteric activation of glutamate dehydrogenase
J. Biol. Chem.
286
37406-37413
2011
Thermus thermophilus, Homo sapiens
Zaganas, I.; Pajecka, K.; Wendel Nielsen, C.; Schousboe, A.; Waagepetersen, H.S.; Plaitakis, A.
The effect of pH and ADP on ammonia affinity for human glutamate dehydrogenases
Metab. Brain Dis.
28
127-131
2013
Homo sapiens
Pajecka, K.; Nielsen, C.W.; Hauge, A.; Zaganas, I.; Bak, L.K.; Schousboe, A.; Plaitakis, A.; Waagepetersen, H.S.
Glutamate dehydrogenase isoforms with N-terminal (His)6- or FLAG-tag retain their kinetic properties and cellular localization
Neurochem. Res.
39
487-499
2014
Homo sapiens (P00367), Homo sapiens (P49448), Homo sapiens, Mus musculus (P26443), Mus musculus
Spanaki, C.; Kotzamani, D.; Plaitakis, A.
Widening spectrum of cellular and subcellular expression of human GLUD1 and GLUD2 glutamate dehydrogenases suggests novel functions
Neurochem. Res.
42
92-107
2017
Homo sapiens (P00367), Homo sapiens (P49448), Homo sapiens
Plaitakis, A.; Kalef-Ezra, E.; Kotzamani, D.; Zaganas, I.; Spanaki, C.
The glutamate dehydrogenase pathway and its roles in cell and tissue biology in health and disease
Biology
6
11
2017
Homo sapiens (P00367), Homo sapiens (P49448), Homo sapiens
Kato, Y.; Kihara, H.; Fukui, K.; Kojima, M.
A ternary complex model of sirtuin4-NAD+-glutamate dehydrogenase
Comput. Biol. Chem.
74
94-104
2018
Homo sapiens (P00367)
Dimovasili, C.; Fadouloglou, V.E.; Kefala, A.; Providaki, M.; Kotsifaki, D.; Kanavouras, K.; Sarrou, I.; Plaitakis, A.; Zaganas, I.; Kokkinidis, M.
Crystal structure of glutamate dehydrogenase 2, a positively selected novel human enzyme involved in brain biology and cancer pathophysiology
J. Neurochem.
157
802-815
2021
Homo sapiens (P00367), Homo sapiens (P49448), Homo sapiens
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