BRENDA - Enzyme Database

Ser95, Asn97, and Thr78 are important for the catalytic function of porcine NADP-dependent isocitrate dehydrogenase

Kim, T.K.; Colman, R.F.; Protein Sci. 14, 140-147 (2005)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
1.1.1.42
expression of wild-type and mutant enzymes in Escherichia coli as maltose-binding fusion proteins
Sus scrofa
Engineering
EC Number
Amino acid exchange
Commentary
Organism
1.1.1.42
N97A
site-directed mutagenesis, decreased Vmax compared to the wild-type enzyme, slightly affected Km values, but increased pKa of the ionizable metal-liganded hydroxyl of enzyme-bound isocitrate compared to the wild-type enzyme
Sus scrofa
1.1.1.42
N97D
site-directed mutagenesis, highly decreased Vmax compared to the wild-type enzyme
Sus scrofa
1.1.1.42
S95A
site-directed mutagenesis, decreased Vmax, and increased Km for isocitrate and Mn2+ compared to the wild-type enzyme
Sus scrofa
1.1.1.42
S95D
site-directed mutagenesis, highly decreased Vmax compared to the wild-type enzyme
Sus scrofa
1.1.1.42
T78A
site-directed mutagenesis, decreased Vmax, and increased Km for isocitrate and Mn2+ compared to the wild-type enzyme
Sus scrofa
1.1.1.42
T78D
site-directed mutagenesis, decreased Vmax compared to the wild-type enzyme
Sus scrofa
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.1.1.42
0.00011
-
Mn2+
recombinant wild-type enzyme, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.0004
-
Mn2+
recombinant mutant N97A, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.0012
-
NADP+
recombinant mutant N97D, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.0024
-
Mn2+
recombinant mutant N97D, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.0033
-
Mn2+
recombinant mutant T78A, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.0049
-
NADP+
recombinant mutant N97A, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.0051
-
NADP+
recombinant mutant S95A, pH 7.4
Sus scrofa
1.1.1.42
0.0053
-
NADP+
recombinant mutant T78A, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.0075
-
isocitrate
recombinant wild-type enzyme, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.0099
-
NADP+
recombinant wild-type enzyme, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.0108
-
NADP+
recombinant mutant T78D, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.0112
-
Mn2+
recombinant mutant S95A, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.014
-
isocitrate
recombinant mutant N97A, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.0237
-
NADP+
recombinant mutant S95D, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.0288
-
Mn2+
recombinant mutant T78D, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.066
-
isocitrate
recombinant mutant T78A, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.0668
-
Mn2+
recombinant mutant S95D, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.072
-
isocitrate
recombinant mutant N97D, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.101
-
isocitrate
recombinant mutant S95A, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.367
-
isocitrate
recombinant mutant T78D, pH 7.4, 25°C
Sus scrofa
1.1.1.42
2.07
-
isocitrate
recombinant mutant S95D, pH 7.4, 25°C
Sus scrofa
Metals/Ions
EC Number
Metals/Ions
Commentary
Organism
Structure
1.1.1.42
Mn2+
as Mn2+-isocitrate complex, Ser95, Asn97, and Thr78 are involved in binding
Sus scrofa
Molecular Weight [Da]
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
1.1.1.42
87000
-
recombinant mutant N97A, native PAGE
Sus scrofa
1.1.1.42
94000
-
recombinant mutant T78A, native PAGE
Sus scrofa
1.1.1.42
97000
-
recombinant mutant N97D, native PAGE
Sus scrofa
1.1.1.42
98000
-
recombinant mutant T78D, native PAGE
Sus scrofa
1.1.1.42
101000
-
recombinant mutant S95A, native PAGE
Sus scrofa
1.1.1.42
104000
-
recombinant wild-type enzyme and mutant S95D, native PAGE
Sus scrofa
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.1.1.42
isocitrate + NADP+
Sus scrofa
-
2-oxoglutarate + CO2 + NADPH
-
-
?
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
1.1.1.42
Sus scrofa
-
-
-
Purification (Commentary)
EC Number
Commentary
Organism
1.1.1.42
recombinant wild-type and mutant enzymes from Escherichia coli, cleavage of the fusion proteins by thrombin, to homogeneity by amylose affinity chromatography
Sus scrofa
Reaction
EC Number
Reaction
Commentary
Organism
1.1.1.42
isocitrate + NADP+ = 2-oxoglutarate + CO2 + NADPH + H+
Ser95, Asn97, and Thr78 are important for the catalysis having distinguishable functions, overview
Sus scrofa
Specific Activity [micromol/min/mg]
EC Number
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
1.1.1.42
0.06
-
purified recombinant mutant S95D
Sus scrofa
1.1.1.42
0.44
-
purified recombinant mutant N97D
Sus scrofa
1.1.1.42
5.29
-
purified recombinant mutant N97A
Sus scrofa
1.1.1.42
6.22
-
purified recombinant mutant T78D
Sus scrofa
1.1.1.42
7.18
-
purified recombinant mutant S95A
Sus scrofa
1.1.1.42
12.7
-
purified recombinant mutant T78A
Sus scrofa
1.1.1.42
35.1
-
purified recombinant wild-type enzyme
Sus scrofa
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.1.1.42
isocitrate + NADP+
-
657309
Sus scrofa
2-oxoglutarate + CO2 + NADPH
-
-
-
?
1.1.1.42
isocitrate + NADP+
Ser95, Asn97, and Thr78 are involved in substrate binding
657309
Sus scrofa
2-oxoglutarate + CO2 + NADPH
-
-
-
?
Subunits
EC Number
Subunits
Commentary
Organism
1.1.1.42
dimer
wild-type and mutant enzymes, circular dichroism and native PAGE
Sus scrofa
Temperature Optimum [°C]
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
1.1.1.42
25
-
assay at
Sus scrofa
pH Optimum
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.1.1.42
7.4
-
assay at
Sus scrofa
pH Range
EC Number
pH Minimum
pH Maximum
Commentary
Organism
1.1.1.42
5
8
pH-profile of wild-type and alanine-substituted mutant enzymes
Sus scrofa
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
1.1.1.42
NADP+
-
Sus scrofa
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
1.1.1.42
expression of wild-type and mutant enzymes in Escherichia coli as maltose-binding fusion proteins
Sus scrofa
Cofactor (protein specific)
EC Number
Cofactor
Commentary
Organism
Structure
1.1.1.42
NADP+
-
Sus scrofa
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
1.1.1.42
N97A
site-directed mutagenesis, decreased Vmax compared to the wild-type enzyme, slightly affected Km values, but increased pKa of the ionizable metal-liganded hydroxyl of enzyme-bound isocitrate compared to the wild-type enzyme
Sus scrofa
1.1.1.42
N97D
site-directed mutagenesis, highly decreased Vmax compared to the wild-type enzyme
Sus scrofa
1.1.1.42
S95A
site-directed mutagenesis, decreased Vmax, and increased Km for isocitrate and Mn2+ compared to the wild-type enzyme
Sus scrofa
1.1.1.42
S95D
site-directed mutagenesis, highly decreased Vmax compared to the wild-type enzyme
Sus scrofa
1.1.1.42
T78A
site-directed mutagenesis, decreased Vmax, and increased Km for isocitrate and Mn2+ compared to the wild-type enzyme
Sus scrofa
1.1.1.42
T78D
site-directed mutagenesis, decreased Vmax compared to the wild-type enzyme
Sus scrofa
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.1.1.42
0.00011
-
Mn2+
recombinant wild-type enzyme, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.0004
-
Mn2+
recombinant mutant N97A, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.0012
-
NADP+
recombinant mutant N97D, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.0024
-
Mn2+
recombinant mutant N97D, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.0033
-
Mn2+
recombinant mutant T78A, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.0049
-
NADP+
recombinant mutant N97A, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.0051
-
NADP+
recombinant mutant S95A, pH 7.4
Sus scrofa
1.1.1.42
0.0053
-
NADP+
recombinant mutant T78A, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.0075
-
isocitrate
recombinant wild-type enzyme, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.0099
-
NADP+
recombinant wild-type enzyme, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.0108
-
NADP+
recombinant mutant T78D, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.0112
-
Mn2+
recombinant mutant S95A, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.014
-
isocitrate
recombinant mutant N97A, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.0237
-
NADP+
recombinant mutant S95D, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.0288
-
Mn2+
recombinant mutant T78D, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.066
-
isocitrate
recombinant mutant T78A, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.0668
-
Mn2+
recombinant mutant S95D, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.072
-
isocitrate
recombinant mutant N97D, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.101
-
isocitrate
recombinant mutant S95A, pH 7.4, 25°C
Sus scrofa
1.1.1.42
0.367
-
isocitrate
recombinant mutant T78D, pH 7.4, 25°C
Sus scrofa
1.1.1.42
2.07
-
isocitrate
recombinant mutant S95D, pH 7.4, 25°C
Sus scrofa
Metals/Ions (protein specific)
EC Number
Metals/Ions
Commentary
Organism
Structure
1.1.1.42
Mn2+
as Mn2+-isocitrate complex, Ser95, Asn97, and Thr78 are involved in binding
Sus scrofa
Molecular Weight [Da] (protein specific)
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
1.1.1.42
87000
-
recombinant mutant N97A, native PAGE
Sus scrofa
1.1.1.42
94000
-
recombinant mutant T78A, native PAGE
Sus scrofa
1.1.1.42
97000
-
recombinant mutant N97D, native PAGE
Sus scrofa
1.1.1.42
98000
-
recombinant mutant T78D, native PAGE
Sus scrofa
1.1.1.42
101000
-
recombinant mutant S95A, native PAGE
Sus scrofa
1.1.1.42
104000
-
recombinant wild-type enzyme and mutant S95D, native PAGE
Sus scrofa
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.1.1.42
isocitrate + NADP+
Sus scrofa
-
2-oxoglutarate + CO2 + NADPH
-
-
?
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
1.1.1.42
recombinant wild-type and mutant enzymes from Escherichia coli, cleavage of the fusion proteins by thrombin, to homogeneity by amylose affinity chromatography
Sus scrofa
Specific Activity [micromol/min/mg] (protein specific)
EC Number
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
1.1.1.42
0.06
-
purified recombinant mutant S95D
Sus scrofa
1.1.1.42
0.44
-
purified recombinant mutant N97D
Sus scrofa
1.1.1.42
5.29
-
purified recombinant mutant N97A
Sus scrofa
1.1.1.42
6.22
-
purified recombinant mutant T78D
Sus scrofa
1.1.1.42
7.18
-
purified recombinant mutant S95A
Sus scrofa
1.1.1.42
12.7
-
purified recombinant mutant T78A
Sus scrofa
1.1.1.42
35.1
-
purified recombinant wild-type enzyme
Sus scrofa
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.1.1.42
isocitrate + NADP+
-
657309
Sus scrofa
2-oxoglutarate + CO2 + NADPH
-
-
-
?
1.1.1.42
isocitrate + NADP+
Ser95, Asn97, and Thr78 are involved in substrate binding
657309
Sus scrofa
2-oxoglutarate + CO2 + NADPH
-
-
-
?
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
1.1.1.42
dimer
wild-type and mutant enzymes, circular dichroism and native PAGE
Sus scrofa
Temperature Optimum [°C] (protein specific)
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
1.1.1.42
25
-
assay at
Sus scrofa
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.1.1.42
7.4
-
assay at
Sus scrofa
pH Range (protein specific)
EC Number
pH Minimum
pH Maximum
Commentary
Organism
1.1.1.42
5
8
pH-profile of wild-type and alanine-substituted mutant enzymes
Sus scrofa