BRENDA - Enzyme Database

Evaluation by mutagenesis of the importance of 3 arginines in alpha, beta, and gamma subunits of human NAD-dependent isocitrate dehydrogenase

Soundar, S.; Park, J.H.; Huh, T.L.; Colman, R.F.; J. Biol. Chem. 278, 52146-52153 (2003)

Data extracted from this reference:

Activating Compound
EC Number
Activating Compound
Commentary
Organism
Structure
1.1.1.41
ADP
allosteric regulation, determination of mol ADP bound to an enzyme tetramer of wild-type and mutant enzymes at different starting concentrations, overview
Homo sapiens
Cloned(Commentary)
EC Number
Commentary
Organism
1.1.1.41
overexpression of the wild-type and a mutant enzyme, the latter containing the wild-type 2 subunits alpha and beta plus an additional third one, gamma, overexpression of the 3-subunit mutant and the same mutant with point mutations of Arg-residues in Escherichia coli
Homo sapiens
Engineering
EC Number
Amino acid exchange
Commentary
Organism
1.1.1.41
R88Q
site-directed mutagenesis, residue of the alpha-subunit, inactive mutant
Homo sapiens
1.1.1.41
R97Q
site-directed mutagenesis, residue of the additional mutant gamma-subunit, highly reduced activity compared to the wild-type enzyme
Homo sapiens
1.1.1.41
R99Q
site-directed mutagenesis, residue of the beta-subunit, reduced activity compared to the wild-type enzyme
Homo sapiens
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.1.1.41
0.004
-
NAD+
purified recombinant mutant gamma-R97Q, pH 7.2, 25°C
Homo sapiens
1.1.1.41
0.014
-
NAD+
purified recombinant mutant beta-R99Q, pH 7.2, 25°C
Homo sapiens
1.1.1.41
0.073
-
NAD+
purified recombinant wild-type enzyme, pH 7.2, 25°C
Homo sapiens
1.1.1.41
0.22
-
Mn2+
purified recombinant wild-type enzyme and mutant beta-R99Q, pH 7.2, 25°C
Homo sapiens
1.1.1.41
0.32
-
isocitrate
purified recombinant wild-type enzyme, pH 7.2, 25°C, in presence of 1 mM ADP
Homo sapiens
1.1.1.41
0.39
-
Mn2+
purified recombinant mutant gamma-R97Q, pH 7.2, 25°C
Homo sapiens
1.1.1.41
0.77
-
isocitrate
purified recombinant mutant beta-R99Q, pH 7.2, 25°C, in presence of 1 mM ADP
Homo sapiens
1.1.1.41
0.9
-
isocitrate
purified recombinant mutant beta-R99Q, pH 7.2, 25°C
Homo sapiens
1.1.1.41
1.8
-
isocitrate
purified recombinant mutant gamma-R97Q, pH 7.2, 25°C, in presence of 1 mM ADP
Homo sapiens
1.1.1.41
2
-
isocitrate
purified recombinant mutant gamma-R97Q, pH 7.2, 25°C
Homo sapiens
1.1.1.41
2.2
-
isocitrate
purified recombinant wild-type enzyme, pH 7.2, 25°C
Homo sapiens
Metals/Ions
EC Number
Metals/Ions
Commentary
Organism
Structure
1.1.1.41
Mn2+
-
Homo sapiens
Molecular Weight [Da]
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
1.1.1.41
315000
-
about, recombinant wild-type and mutant enzymes, gel filtration
Homo sapiens
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.1.1.41
threo-Ds-isocitrate + NAD+
Homo sapiens
-
2-oxoglutarate + CO2 + NADH
-
-
?
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
1.1.1.41
Homo sapiens
-
-
-
Purification (Commentary)
EC Number
Commentary
Organism
1.1.1.41
recombinant wild-type and mutant enzymes from Escherichia coli
Homo sapiens
Reaction
EC Number
Reaction
Commentary
Organism
1.1.1.41
isocitrate + NAD+ = 2-oxoglutarate + CO2 + NADH
Arg97, and Arg99 contribute to activity, Arg88 is essential for catalysis
Homo sapiens
Specific Activity [micromol/min/mg]
EC Number
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
1.1.1.41
2.2
-
purified recombinant mutant gamma-R97Q
Homo sapiens
1.1.1.41
13.5
-
purified recombinant mutant beta-R99Q
Homo sapiens
1.1.1.41
21.7
-
purified recombinant wild-type enzyme
Homo sapiens
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.1.1.41
threo-Ds-isocitrate + NAD+
-
656189
Homo sapiens
2-oxoglutarate + CO2 + NADH
-
-
-
?
Subunits
EC Number
Subunits
Commentary
Organism
1.1.1.41
More
enzyme subunit ratio of recombinant wild-type and mutant enzymes, overview
Homo sapiens
Temperature Optimum [°C]
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
1.1.1.41
25
-
assay at
Homo sapiens
pH Optimum
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.1.1.41
7.2
-
assay at
Homo sapiens
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
1.1.1.41
NAD+
specific for
Homo sapiens
Activating Compound (protein specific)
EC Number
Activating Compound
Commentary
Organism
Structure
1.1.1.41
ADP
allosteric regulation, determination of mol ADP bound to an enzyme tetramer of wild-type and mutant enzymes at different starting concentrations, overview
Homo sapiens
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
1.1.1.41
overexpression of the wild-type and a mutant enzyme, the latter containing the wild-type 2 subunits alpha and beta plus an additional third one, gamma, overexpression of the 3-subunit mutant and the same mutant with point mutations of Arg-residues in Escherichia coli
Homo sapiens
Cofactor (protein specific)
EC Number
Cofactor
Commentary
Organism
Structure
1.1.1.41
NAD+
specific for
Homo sapiens
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
1.1.1.41
R88Q
site-directed mutagenesis, residue of the alpha-subunit, inactive mutant
Homo sapiens
1.1.1.41
R97Q
site-directed mutagenesis, residue of the additional mutant gamma-subunit, highly reduced activity compared to the wild-type enzyme
Homo sapiens
1.1.1.41
R99Q
site-directed mutagenesis, residue of the beta-subunit, reduced activity compared to the wild-type enzyme
Homo sapiens
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.1.1.41
0.004
-
NAD+
purified recombinant mutant gamma-R97Q, pH 7.2, 25°C
Homo sapiens
1.1.1.41
0.014
-
NAD+
purified recombinant mutant beta-R99Q, pH 7.2, 25°C
Homo sapiens
1.1.1.41
0.073
-
NAD+
purified recombinant wild-type enzyme, pH 7.2, 25°C
Homo sapiens
1.1.1.41
0.22
-
Mn2+
purified recombinant wild-type enzyme and mutant beta-R99Q, pH 7.2, 25°C
Homo sapiens
1.1.1.41
0.32
-
isocitrate
purified recombinant wild-type enzyme, pH 7.2, 25°C, in presence of 1 mM ADP
Homo sapiens
1.1.1.41
0.39
-
Mn2+
purified recombinant mutant gamma-R97Q, pH 7.2, 25°C
Homo sapiens
1.1.1.41
0.77
-
isocitrate
purified recombinant mutant beta-R99Q, pH 7.2, 25°C, in presence of 1 mM ADP
Homo sapiens
1.1.1.41
0.9
-
isocitrate
purified recombinant mutant beta-R99Q, pH 7.2, 25°C
Homo sapiens
1.1.1.41
1.8
-
isocitrate
purified recombinant mutant gamma-R97Q, pH 7.2, 25°C, in presence of 1 mM ADP
Homo sapiens
1.1.1.41
2
-
isocitrate
purified recombinant mutant gamma-R97Q, pH 7.2, 25°C
Homo sapiens
1.1.1.41
2.2
-
isocitrate
purified recombinant wild-type enzyme, pH 7.2, 25°C
Homo sapiens
Metals/Ions (protein specific)
EC Number
Metals/Ions
Commentary
Organism
Structure
1.1.1.41
Mn2+
-
Homo sapiens
Molecular Weight [Da] (protein specific)
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
1.1.1.41
315000
-
about, recombinant wild-type and mutant enzymes, gel filtration
Homo sapiens
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.1.1.41
threo-Ds-isocitrate + NAD+
Homo sapiens
-
2-oxoglutarate + CO2 + NADH
-
-
?
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
1.1.1.41
recombinant wild-type and mutant enzymes from Escherichia coli
Homo sapiens
Specific Activity [micromol/min/mg] (protein specific)
EC Number
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
1.1.1.41
2.2
-
purified recombinant mutant gamma-R97Q
Homo sapiens
1.1.1.41
13.5
-
purified recombinant mutant beta-R99Q
Homo sapiens
1.1.1.41
21.7
-
purified recombinant wild-type enzyme
Homo sapiens
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.1.1.41
threo-Ds-isocitrate + NAD+
-
656189
Homo sapiens
2-oxoglutarate + CO2 + NADH
-
-
-
?
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
1.1.1.41
More
enzyme subunit ratio of recombinant wild-type and mutant enzymes, overview
Homo sapiens
Temperature Optimum [°C] (protein specific)
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
1.1.1.41
25
-
assay at
Homo sapiens
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.1.1.41
7.2
-
assay at
Homo sapiens