BRENDA - Enzyme Database

Implication by site-directed mutagenesis of Arg314 and Tyr316 in the coenzyme site of pig mitochondrial NADP-dependent isocitrate dehydrogenase

Lee, P.; Colman, R.F.; Arch. Biochem. Biophys. 401, 81-90 (2002)

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 TB1 as maltose binding fusion proteins
Sus scrofa
Engineering
EC Number
Amino acid exchange
Commentary
Organism
1.1.1.42
K321Q
site-directed mutagenesis, kinetics are similar to the wild-type enzyme
Sus scrofa
1.1.1.42
R314Q
site-directed mutagenesis, 10fold increased Km for NADP+ compared to the wild-type enzyme
Sus scrofa
1.1.1.42
R323Q
site-directed mutagenesis, kinetics are similar to the wild-type enzyme
Sus scrofa
1.1.1.42
Y316F
site-directed mutagenesis, kinetics are similar to the wild-type enzyme
Sus scrofa
1.1.1.42
Y316L
site-directed mutagenesis, 4fold increased Km for NADP+ compared to the wild-type enzyme
Sus scrofa
Inhibitors
EC Number
Inhibitors
Commentary
Organism
Structure
1.1.1.42
NAD+
-
Sus scrofa
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.1.1.42
0.0003
-
Mn2+
pH 7.4, recombinant wild-type enzyme
Sus scrofa
1.1.1.42
0.0004
-
Mn2+
pH 7.4, recombinant mutant Y316F and K323Q
Sus scrofa
1.1.1.42
0.0005
-
Mn2+
pH 7.4, recombinant mutant K321Q
Sus scrofa
1.1.1.42
0.0059
-
isocitrate
pH 7.4, recombinant mutant Y316F
Sus scrofa
1.1.1.42
0.0062
-
isocitrate
pH 7.4, recombinant mutant K323Q
Sus scrofa
1.1.1.42
0.0074
-
isocitrate
pH 7.4, recombinant wild-type enzyme
Sus scrofa
1.1.1.42
0.0077
-
NADP+
pH 7.4, recombinant mutant K323Q
Sus scrofa
1.1.1.42
0.008
-
NADP+
pH 7.4, recombinant mutant Y316F and K321Q
Sus scrofa
1.1.1.42
0.009
-
NADP+
pH 7.4, recombinant wild-type enzyme
Sus scrofa
1.1.1.42
0.01
-
isocitrate
pH 7.4, recombinant mutant K321Q
Sus scrofa
1.1.1.42
0.032
-
NADP+
pH 7.4, recombinant mutant Y316L
Sus scrofa
1.1.1.42
0.092
-
NADP+
pH 7.4, recombinant mutant R314Q
Sus scrofa
Localization
EC Number
Localization
Commentary
Organism
GeneOntology No.
Textmining
1.1.1.42
mitochondrion
-
Sus scrofa
5739
-
Metals/Ions
EC Number
Metals/Ions
Commentary
Organism
Structure
1.1.1.42
Mn2+
-
Sus scrofa
Molecular Weight [Da]
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
1.1.1.42
82000
-
recombinant mutant Y316F, dynamic light scattering
Sus scrofa
1.1.1.42
86000
-
recombinant wild-type enzyme, dynamic light scattering
Sus scrofa
1.1.1.42
87000
-
recombinant mutants K321Q and R314Q, dynamic light scattering
Sus scrofa
1.1.1.42
97900
-
recombinant mutant Y316L, native PAGE at pH 5.5
Sus scrofa
1.1.1.42
99800
-
recombinant wild-type enzyme, native PAGE at pH 5.5
Sus scrofa
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, to homogeneity by amylose affinity chromatography, thrombin digestion, and DEAE ion exchange chromatography
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
7
-
purified recombinant mutant Y316L
Sus scrofa
1.1.1.42
18.1
-
purified recombinant mutant R314Q
Sus scrofa
1.1.1.42
38.6
-
purified recombinant wild-type enzyme
Sus scrofa
1.1.1.42
38.7
-
purified recombinant mutant K323Q
Sus scrofa
1.1.1.42
39.6
-
purified recombinant mutant K321Q
Sus scrofa
1.1.1.42
40.3
-
purified recombinant mutant Y316F
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+
-
654341
Sus scrofa
2-oxoglutarate + CO2 + NADPH
-
-
-
?
Subunits
EC Number
Subunits
Commentary
Organism
1.1.1.42
dimer
residues Arg314, Tyr316, Lys321, and Arg323 are not involved in subunit interaction
Sus scrofa
pH Optimum
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.1.1.42
7.4
-
assay at
Sus scrofa
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
1.1.1.42
NADP+
Arg314 and the aromatic ring of Tyr316 are important for affinity to the cofactor and catalysis, interaction with 2'-phosphate group of NADP+
Sus scrofa
Ki Value [mM]
EC Number
Ki Value [mM]
Ki Value maximum [mM]
Inhibitor
Commentary
Organism
Structure
1.1.1.42
8
-
NAD+
pH 7.4, versus NADPH, recombinant mutant R314Q
Sus scrofa
1.1.1.42
11
-
NAD+
pH 7.4, versus NADPH, recombinant wild-type enzyme and mutant Y316L
Sus scrofa
1.1.1.42
12
-
NAD+
pH 7.4, versus NADPH, recombinant mutant Y316F
Sus scrofa
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
1.1.1.42
expression of wild-type and mutant enzymes in Escherichia coli TB1 as maltose binding fusion proteins
Sus scrofa
Cofactor (protein specific)
EC Number
Cofactor
Commentary
Organism
Structure
1.1.1.42
NADP+
Arg314 and the aromatic ring of Tyr316 are important for affinity to the cofactor and catalysis, interaction with 2'-phosphate group of NADP+
Sus scrofa
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
1.1.1.42
K321Q
site-directed mutagenesis, kinetics are similar to the wild-type enzyme
Sus scrofa
1.1.1.42
R314Q
site-directed mutagenesis, 10fold increased Km for NADP+ compared to the wild-type enzyme
Sus scrofa
1.1.1.42
R323Q
site-directed mutagenesis, kinetics are similar to the wild-type enzyme
Sus scrofa
1.1.1.42
Y316F
site-directed mutagenesis, kinetics are similar to the wild-type enzyme
Sus scrofa
1.1.1.42
Y316L
site-directed mutagenesis, 4fold increased Km for NADP+ compared to the wild-type enzyme
Sus scrofa
Inhibitors (protein specific)
EC Number
Inhibitors
Commentary
Organism
Structure
1.1.1.42
NAD+
-
Sus scrofa
Ki Value [mM] (protein specific)
EC Number
Ki Value [mM]
Ki Value maximum [mM]
Inhibitor
Commentary
Organism
Structure
1.1.1.42
8
-
NAD+
pH 7.4, versus NADPH, recombinant mutant R314Q
Sus scrofa
1.1.1.42
11
-
NAD+
pH 7.4, versus NADPH, recombinant wild-type enzyme and mutant Y316L
Sus scrofa
1.1.1.42
12
-
NAD+
pH 7.4, versus NADPH, recombinant mutant Y316F
Sus scrofa
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.1.1.42
0.0003
-
Mn2+
pH 7.4, recombinant wild-type enzyme
Sus scrofa
1.1.1.42
0.0004
-
Mn2+
pH 7.4, recombinant mutant Y316F and K323Q
Sus scrofa
1.1.1.42
0.0005
-
Mn2+
pH 7.4, recombinant mutant K321Q
Sus scrofa
1.1.1.42
0.0059
-
isocitrate
pH 7.4, recombinant mutant Y316F
Sus scrofa
1.1.1.42
0.0062
-
isocitrate
pH 7.4, recombinant mutant K323Q
Sus scrofa
1.1.1.42
0.0074
-
isocitrate
pH 7.4, recombinant wild-type enzyme
Sus scrofa
1.1.1.42
0.0077
-
NADP+
pH 7.4, recombinant mutant K323Q
Sus scrofa
1.1.1.42
0.008
-
NADP+
pH 7.4, recombinant mutant Y316F and K321Q
Sus scrofa
1.1.1.42
0.009
-
NADP+
pH 7.4, recombinant wild-type enzyme
Sus scrofa
1.1.1.42
0.01
-
isocitrate
pH 7.4, recombinant mutant K321Q
Sus scrofa
1.1.1.42
0.032
-
NADP+
pH 7.4, recombinant mutant Y316L
Sus scrofa
1.1.1.42
0.092
-
NADP+
pH 7.4, recombinant mutant R314Q
Sus scrofa
Localization (protein specific)
EC Number
Localization
Commentary
Organism
GeneOntology No.
Textmining
1.1.1.42
mitochondrion
-
Sus scrofa
5739
-
Metals/Ions (protein specific)
EC Number
Metals/Ions
Commentary
Organism
Structure
1.1.1.42
Mn2+
-
Sus scrofa
Molecular Weight [Da] (protein specific)
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
1.1.1.42
82000
-
recombinant mutant Y316F, dynamic light scattering
Sus scrofa
1.1.1.42
86000
-
recombinant wild-type enzyme, dynamic light scattering
Sus scrofa
1.1.1.42
87000
-
recombinant mutants K321Q and R314Q, dynamic light scattering
Sus scrofa
1.1.1.42
97900
-
recombinant mutant Y316L, native PAGE at pH 5.5
Sus scrofa
1.1.1.42
99800
-
recombinant wild-type enzyme, native PAGE at pH 5.5
Sus scrofa
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
1.1.1.42
recombinant wild-type and mutant enzymes from Escherichia coli, to homogeneity by amylose affinity chromatography, thrombin digestion, and DEAE ion exchange 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
7
-
purified recombinant mutant Y316L
Sus scrofa
1.1.1.42
18.1
-
purified recombinant mutant R314Q
Sus scrofa
1.1.1.42
38.6
-
purified recombinant wild-type enzyme
Sus scrofa
1.1.1.42
38.7
-
purified recombinant mutant K323Q
Sus scrofa
1.1.1.42
39.6
-
purified recombinant mutant K321Q
Sus scrofa
1.1.1.42
40.3
-
purified recombinant mutant Y316F
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+
-
654341
Sus scrofa
2-oxoglutarate + CO2 + NADPH
-
-
-
?
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
1.1.1.42
dimer
residues Arg314, Tyr316, Lys321, and Arg323 are not involved in subunit interaction
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