BRENDA - Enzyme Database

Evaluation by mutagenesis of the roles of His309, His315, and His319 in the coenzyme site of pig heart NADP-dependent isocitrate dehydrogenase

Huang, Y.C.; Colman, R.F.; Biochemistry 41, 5637-5643 (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 as maltose binding fusion proteins
Sus scrofa
Engineering
EC Number
Amino acid exchange
Commentary
Organism
1.1.1.42
H309F
site-directed mutagenesis, inactive mutant, poor cofactor binding, altered secondary structure
Sus scrofa
1.1.1.42
H309Q
site-directed mutagenesis, inactive mutant, poor cofactor binding, altered secondary structure
Sus scrofa
1.1.1.42
H315Q
site-directed mutagenesis, 40fold increased Km for NADP+ compared to the wild-type enzyme
Sus scrofa
1.1.1.42
H319Q
site-directed mutagenesis, cofactor binding and kinetics similar to the wild-type enzyme, slightly reduced activity
Sus scrofa
Inhibitors
EC Number
Inhibitors
Commentary
Organism
Structure
1.1.1.42
NADPH
-
Sus scrofa
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.1.1.42
additional information
-
additional information
measurement methods, the maltose binding fusion protein of the recombinant enzymes alters the kinetic parameters, overview
Sus scrofa
1.1.1.42
0.00033
-
Mn2+
pH 7.4, 25C, recombinant wild-type
Sus scrofa
1.1.1.42
0.00039
-
Mn2+
pH 7.4, 25C, recombinant mutant H319Q
Sus scrofa
1.1.1.42
0.0027
-
Mn2+
pH 7.4, 25C, recombinant mutant H315Q
Sus scrofa
1.1.1.42
0.0051
-
NADP+
pH 7.4, 25C, recombinant mutant H319Q
Sus scrofa
1.1.1.42
0.0056
-
NADP+
pH 7.4, 25C, recombinant wild-type
Sus scrofa
1.1.1.42
0.0083
-
DL-isocitrate
pH 7.4, 25C, recombinant mutant H319Q
Sus scrofa
1.1.1.42
0.0084
-
DL-isocitrate
pH 7.4, 25C, recombinant wild-type
Sus scrofa
1.1.1.42
0.0087
-
DL-isocitrate
pH 7.4, 25C, recombinant mutant H315Q
Sus scrofa
1.1.1.42
0.218
-
NADP+
pH 7.4, 25C, recombinant mutant H315Q
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+
required, not bound normally
Sus scrofa
Molecular Weight [Da]
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
1.1.1.42
46600
-
2 * 46600, recombinant thrombin cleaved wild-type and mutant enzymes, SDS-PAGE
Sus scrofa
1.1.1.42
90000
-
recombinant maltose binding fusion proteins, wild-type and mutant enzymes, gel filtration
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
DL-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 maltose binding fusion proteins from Escherichia coli by amylose affinity and size exclusion chromatography
Sus scrofa
Source Tissue
EC Number
Source Tissue
Commentary
Organism
Textmining
1.1.1.42
heart
-
Sus scrofa
-
Specific Activity [micromol/min/mg]
EC Number
Specific Activity Minimum [mol/min/mg]
Specific Activity Maximum [mol/min/mg]
Commentary
Organism
1.1.1.42
4.1
-
purified recombinant mutant H315Q
Sus scrofa
1.1.1.42
20.1
-
purified recombinant mutant H319Q
Sus scrofa
1.1.1.42
37.8
-
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
DL-isocitrate + NADP+
-
654649
Sus scrofa
2-oxoglutarate + CO2 + NADPH
-
-
-
?
1.1.1.42
Ds-isocitrate + NADP+
-
654649
Sus scrofa
2-oxoglutarate + CO2 + NADPH
-
-
-
?
1.1.1.42
additional information
His319 and His315 are not responsible for enzyme Fe2+-isocitrate cleavage
654649
Sus scrofa
?
-
-
-
-
Subunits
EC Number
Subunits
Commentary
Organism
1.1.1.42
dimer
2 * 46600, recombinant thrombin cleaved wild-type and mutant enzymes, SDS-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
-
Sus scrofa
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
1.1.1.42
NADP+
binding of 1 mol NADP+ per enzyme subunit, His309 is highly important for cofactor binding, HIs315 is involved, while His319 is not
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+
binding of 1 mol NADP+ per enzyme subunit, His309 is highly important for cofactor binding, HIs315 is involved, while His319 is not
Sus scrofa
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
1.1.1.42
H309F
site-directed mutagenesis, inactive mutant, poor cofactor binding, altered secondary structure
Sus scrofa
1.1.1.42
H309Q
site-directed mutagenesis, inactive mutant, poor cofactor binding, altered secondary structure
Sus scrofa
1.1.1.42
H315Q
site-directed mutagenesis, 40fold increased Km for NADP+ compared to the wild-type enzyme
Sus scrofa
1.1.1.42
H319Q
site-directed mutagenesis, cofactor binding and kinetics similar to the wild-type enzyme, slightly reduced activity
Sus scrofa
Inhibitors (protein specific)
EC Number
Inhibitors
Commentary
Organism
Structure
1.1.1.42
NADPH
-
Sus scrofa
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.1.1.42
additional information
-
additional information
measurement methods, the maltose binding fusion protein of the recombinant enzymes alters the kinetic parameters, overview
Sus scrofa
1.1.1.42
0.00033
-
Mn2+
pH 7.4, 25C, recombinant wild-type
Sus scrofa
1.1.1.42
0.00039
-
Mn2+
pH 7.4, 25C, recombinant mutant H319Q
Sus scrofa
1.1.1.42
0.0027
-
Mn2+
pH 7.4, 25C, recombinant mutant H315Q
Sus scrofa
1.1.1.42
0.0051
-
NADP+
pH 7.4, 25C, recombinant mutant H319Q
Sus scrofa
1.1.1.42
0.0056
-
NADP+
pH 7.4, 25C, recombinant wild-type
Sus scrofa
1.1.1.42
0.0083
-
DL-isocitrate
pH 7.4, 25C, recombinant mutant H319Q
Sus scrofa
1.1.1.42
0.0084
-
DL-isocitrate
pH 7.4, 25C, recombinant wild-type
Sus scrofa
1.1.1.42
0.0087
-
DL-isocitrate
pH 7.4, 25C, recombinant mutant H315Q
Sus scrofa
1.1.1.42
0.218
-
NADP+
pH 7.4, 25C, recombinant mutant H315Q
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+
required, not bound normally
Sus scrofa
Molecular Weight [Da] (protein specific)
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
1.1.1.42
46600
-
2 * 46600, recombinant thrombin cleaved wild-type and mutant enzymes, SDS-PAGE
Sus scrofa
1.1.1.42
90000
-
recombinant maltose binding fusion proteins, wild-type and mutant enzymes, gel filtration
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
DL-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 maltose binding fusion proteins from Escherichia coli by amylose affinity and size exclusion chromatography
Sus scrofa
Source Tissue (protein specific)
EC Number
Source Tissue
Commentary
Organism
Textmining
1.1.1.42
heart
-
Sus scrofa
-
Specific Activity [micromol/min/mg] (protein specific)
EC Number
Specific Activity Minimum [mol/min/mg]
Specific Activity Maximum [mol/min/mg]
Commentary
Organism
1.1.1.42
4.1
-
purified recombinant mutant H315Q
Sus scrofa
1.1.1.42
20.1
-
purified recombinant mutant H319Q
Sus scrofa
1.1.1.42
37.8
-
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
DL-isocitrate + NADP+
-
654649
Sus scrofa
2-oxoglutarate + CO2 + NADPH
-
-
-
?
1.1.1.42
Ds-isocitrate + NADP+
-
654649
Sus scrofa
2-oxoglutarate + CO2 + NADPH
-
-
-
?
1.1.1.42
additional information
His319 and His315 are not responsible for enzyme Fe2+-isocitrate cleavage
654649
Sus scrofa
?
-
-
-
-
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
1.1.1.42
dimer
2 * 46600, recombinant thrombin cleaved wild-type and mutant enzymes, SDS-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
-
Sus scrofa