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Literature summary for 1.4.1.2 extracted from

  • Griffin, J.; Engel, P.C.
    An examination by site-directed mutagenesis of putative key residues in the determination of coenzyme specificity in clostridial NAD-dependent glutamate dehydrogenase (2011), Enzyme Res., 2011, 595793.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
cloning and expression of soluble wild-type and mutant enzymes in Escherichia coli strain XL1-Blue [Clostridium] symbiosum

Protein Variants

Protein Variants Comment Organism
A242G site-directed mutagenesis, the mutant shows A242G showed a decreased overall catalytic efficiency for NADH at all pH values of pH 6.0-8.0 after Ala replacement with Gly compared to the wild-type enzyme, the mutation had a severe effect on the overall catalytic efficiency with NADPH as coenzyme [Clostridium] symbiosum
D263K site-directed mutagenesis, the D263K mutation produces remarkably little change in the kinetic parameters for NADH at pH 6.0-8.0 compared to the wild-type enzyme, with NADPH at all three pH values the kcat for the mutant is much higher than for wild-type GDH, and this factor increases from pH 6.0 to pH 7.0 and pH 8.0 [Clostridium] symbiosum
F238S site-directed mutagenesis, the mutant shows markedly increased catalytic efficiency with NADPH, especially at pH 8.0 in the range of pH 6.0-8.0 [Clostridium] symbiosum
F238S/P262S site-directed mutagenesis, the mutant shows markedly increased catalytic efficiency with NADPH, especially at pH 8.0 in the range of pH 6.0-8.0 [Clostridium] symbiosum
P262S site-directed mutagenesis, the mutant shows markedly increased catalytic efficiency with NADPH, especially at pH 8.0 in the range of pH 6.0-8.0 [Clostridium] symbiosum

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information enzyme kinetics of wild-tyype and mutant enzymes with NADPH, overview [Clostridium] symbiosum
0.018
-
NADH pH 6.0, 25°C, mutant P262S [Clostridium] symbiosum
0.02
-
NADH pH 7.0, 25°C, recombinant wild-type enzyme [Clostridium] symbiosum
0.022
-
NADH pH 6.0, 25°C, mutant D263K [Clostridium] symbiosum
0.023
-
NADH pH 7.0, 25°C, mutant D263K [Clostridium] symbiosum
0.025
-
NADH pH 7.0, 25°C, mutant A242G [Clostridium] symbiosum
0.028
-
NADH pH 6.0, 25°C, mutant F238S [Clostridium] symbiosum
0.034
-
NADH pH 6.0, 25°C, mutant A242G [Clostridium] symbiosum
0.035
-
NADH pH 7.0, 25°C, mutant F238S/P262S [Clostridium] symbiosum
0.036
-
NADH pH 6.0, 25°C, recombinant wild-type enzyme [Clostridium] symbiosum
0.04
-
NADH pH 7.0, 25°C, mutant F238S [Clostridium] symbiosum
0.047
-
NADH pH 7.0, 25°C, mutant P262S [Clostridium] symbiosum
0.048
-
NADH pH 6.0, 25°C, mutant F238S/P262S [Clostridium] symbiosum
0.078
-
NADH pH 8.0, 25°C, recombinant wild-type enzyme [Clostridium] symbiosum
0.085
-
NADH pH 8.0, 25°C, mutant A242G [Clostridium] symbiosum
0.091
-
NADH pH 8.0, 25°C, mutant D263K [Clostridium] symbiosum
0.142
-
NADH pH 8.0, 25°C, mutant P262S [Clostridium] symbiosum
0.204
-
NADH pH 8.0, 25°C, mutant F238S/P262S [Clostridium] symbiosum
0.23
-
NADH pH 8.0, 25°C, mutant F238S [Clostridium] symbiosum

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
L-glutamate + H2O + NAD+ [Clostridium] symbiosum
-
2-oxoglutarate + NH3 + NADH + H+
-
r

Organism

Organism UniProt Comment Textmining
[Clostridium] symbiosum
-
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant wild-type and mutant enzymes from Escherichia coli strain XL1-Blue [Clostridium] symbiosum

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
L-glutamate + H2O + NAD+
-
[Clostridium] symbiosum 2-oxoglutarate + NH3 + NADH + H+
-
r

Synonyms

Synonyms Comment Organism
NAD+-specific GDH
-
[Clostridium] symbiosum
NAD-dependent glutamate dehydrogenase
-
[Clostridium] symbiosum

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at [Clostridium] symbiosum

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
11.2
-
NADH pH 6.0, 25°C, mutant A242G [Clostridium] symbiosum
13.6
-
NADH pH 6.0, 25°C, mutant F238S [Clostridium] symbiosum
13.9
-
NADH pH 6.0, 25°C, mutant F238S/P262S [Clostridium] symbiosum
14.4
-
NADH pH 6.0, 25°C, mutant P262S [Clostridium] symbiosum
14.6
-
NADH pH 6.0, 25°C, mutant D263K [Clostridium] symbiosum
19.9
-
NADH pH 6.0, 25°C, recombinant wild-type enzyme [Clostridium] symbiosum
65.1
-
NADH pH 7.0, 25°C, mutant A242G [Clostridium] symbiosum
80
-
NADH pH 7.0, 25°C, mutant D263K [Clostridium] symbiosum
105
-
NADH pH 7.0, 25°C, mutant F238S/P262S [Clostridium] symbiosum
130.2
-
NADH pH 7.0, 25°C, recombinant wild-type enzyme [Clostridium] symbiosum
133
-
NADH pH 8.0, 25°C, mutant F238S/P262S [Clostridium] symbiosum
139
-
NADH pH 7.0, 25°C, mutant F238S [Clostridium] symbiosum
150
-
NADH pH 8.0, 25°C, mutant D263K [Clostridium] symbiosum
160
-
NADH pH 7.0, 25°C, mutant P262S [Clostridium] symbiosum
177
-
NADH pH 8.0, 25°C, mutant A242G [Clostridium] symbiosum
232
-
NADH pH 8.0, 25°C, recombinant wild-type enzyme [Clostridium] symbiosum
402
-
NADH pH 8.0, 25°C, mutant F238S [Clostridium] symbiosum
490
-
NADH pH 8.0, 25°C, mutant P262S [Clostridium] symbiosum

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
6 8 assay at [Clostridium] symbiosum

Cofactor

Cofactor Comment Organism Structure
additional information in the wild-type enzyme there is a strong pH dependence in the level of discrimination between NADH and NADPH [Clostridium] symbiosum
NAD+ the core fingerprint for coenzyme binding is the conserved sequence Gly-X-Gly-X-X-Gly, which consists of residues that form a hydrophobic core between the beta-strands and the alpha-helix, and glycine residues that allow for a tight turn between the first beta-strand and the following alpha-helix. In all these enzymes, the last residue in helix alphaA has to be a Gly, the first Gly of the consensus, because any larger residue would interfere with the adenine ribose. Coenzyme binding structure, overview [Clostridium] symbiosum
NADH the core fingerprint for coenzyme binding is the conserved sequence Gly-X-Gly-X-X-Gly, which consists of residues that form a hydrophobic core between the beta-strands and the alpha-helix, and glycine residues that allow for a tight turn between the first beta-strand and the following alpha-helix. In all these enzymes, the last residue in helix alphaA has to be a Gly, the first Gly of the consensus, because any larger residue would interfere with the adenine ribose. Coenzyme binding structure, overview [Clostridium] symbiosum

General Information

General Information Comment Organism
additional information at position 242, P6 of the core fingerprint, where NAD+- and NADP+-dependent enzymes normally have Gly or Ala, respectively, clostridial GDH already has Ala. Replacement with Gly produced negligible shift in coenzyme specificity [Clostridium] symbiosum

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
289
-
NADH pH 6.0, 25°C, mutant F238S/P262S [Clostridium] symbiosum
329
-
NADH pH 6.0, 25°C, mutant A242G [Clostridium] symbiosum
485
-
NADH pH 6.0, 25°C, mutant F238S [Clostridium] symbiosum
553
-
NADH pH 6.0, 25°C, recombinant wild-type enzyme [Clostridium] symbiosum
655
-
NADH pH 8.0, 25°C, mutant F238S/P262S [Clostridium] symbiosum
663
-
NADH pH 6.0, 25°C, mutant D263K [Clostridium] symbiosum
800
-
NADH pH 6.0, 25°C, mutant P262S [Clostridium] symbiosum
1648
-
NADH pH 8.0, 25°C, mutant D263K [Clostridium] symbiosum
1747
-
NADH pH 8.0, 25°C, mutant F238S [Clostridium] symbiosum
2082
-
NADH pH 8.0, 25°C, mutant A242G [Clostridium] symbiosum
2604
-
NADH pH 7.0, 25°C, mutant A242G [Clostridium] symbiosum
2974
-
NADH pH 8.0, 25°C, recombinant wild-type enzyme [Clostridium] symbiosum
3000
-
NADH pH 7.0, 25°C, mutant F238S/P262S [Clostridium] symbiosum
3404
-
NADH pH 7.0, 25°C, mutant P262S [Clostridium] symbiosum
3450
-
NADH pH 8.0, 25°C, mutant P262S [Clostridium] symbiosum
3475
-
NADH pH 7.0, 25°C, mutant F238S [Clostridium] symbiosum
3478
-
NADH pH 7.0, 25°C, mutant D263K [Clostridium] symbiosum
6500
-
NADH pH 7.0, 25°C, recombinant wild-type enzyme [Clostridium] symbiosum