BRENDA - Enzyme Database

Critical role of Lys212 and Tyr140 in porcine NADP-dependent isocitrate dehydrogenase

Kim, T.K.; Lee, P.; Colman, R.F.; J. Biol. Chem. 278, 49323-49331 (2003)

Data extracted from this reference:

Activating Compound
EC Number
Activating Compound
Commentary
Organism
Structure
1.1.1.42
acetic acid
mutant Y140T, 106fold activation compared to the wild-type enzyme
Sus scrofa
1.1.1.42
ethylamine
mutant K212Q, 4fold activation compared to the wild-type enzyme
Sus scrofa
1.1.1.42
phenol
mutant Y140T, 88fold activation compared to the wild-type enzyme
Sus scrofa
Cloned(Commentary)
EC Number
Commentary
Organism
1.1.1.42
expression of wild-type and mutant enzymes in Escherichia coli
Sus scrofa
Engineering
EC Number
Amino acid exchange
Commentary
Organism
1.1.1.42
K212Q
site-directed mutagenesis, highly decreased activity in both reaction directions compared to the wild-type enzyme, altered pH-dependency of the activity
Sus scrofa
1.1.1.42
K212R
site-directed mutagenesis, highly decreased activity in both reaction directions compared to the wild-type enzyme, altered pH-dependency of the activity
Sus scrofa
1.1.1.42
K212Y
site-directed mutagenesis, highly decreased activity in both reaction directions compared to the wild-type enzyme, altered pH-dependency of the activity
Sus scrofa
1.1.1.42
Y140E
site-directed mutagenesis, highly decreased activity in both reaction directions compared to the wild-type enzyme, unaltered Km for isocitrate and NADP+
Sus scrofa
1.1.1.42
Y140F
site-directed mutagenesis, highly decreased activity in both reaction directions compared to the wild-type enzyme, unaltered Km for isocitrate and NADP+
Sus scrofa
1.1.1.42
Y140K
site-directed mutagenesis, highly decreased activity in both reaction directions compared to the wild-type enzyme, unaltered Km for isocitrate and NADP+
Sus scrofa
1.1.1.42
Y140T
site-directed mutagenesis, highly decreased activity in both reaction directions compared to the wild-type enzyme, unaltered Km for isocitrate and NADP+, highly increased activation by added exogenous acetic acid and phenol 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
0.00004
-
Mn2+
pH 7.4, mutant Y140F
Sus scrofa
1.1.1.42
0.000084
-
Mn2+
pH 7.4, mutant Y140E
Sus scrofa
1.1.1.42
0.00011
-
Mn2+
pH 7.4, wild-type enzyme
Sus scrofa
1.1.1.42
0.00042
-
NADP+
pH 7.4, mutant Y140E
Sus scrofa
1.1.1.42
0.00046
-
NADP+
pH 7.4, mutant Y140T
Sus scrofa
1.1.1.42
0.00051
-
NADP+
pH 7.4, mutant Y140K
Sus scrofa
1.1.1.42
0.0006
-
isocitrate
pH 7.4, mutant Y140T
Sus scrofa
1.1.1.42
0.0007
-
isocitrate
pH 7.4, mutant Y140E
Sus scrofa
1.1.1.42
0.00091
-
Mn2+
pH 7.4, mutant K212Q
Sus scrofa
1.1.1.42
0.0011
-
Mn2+
pH 7.4, mutant K212Y
Sus scrofa
1.1.1.42
0.0023
-
NADP+
pH 7.4, mutant Y140F
Sus scrofa
1.1.1.42
0.0052
-
isocitrate
pH 7.4, mutant Y140F
Sus scrofa
1.1.1.42
0.0053
-
isocitrate
pH 7.4, mutant Y140K
Sus scrofa
1.1.1.42
0.0073
-
NADP+
pH 7.4, mutant K212Y
Sus scrofa
1.1.1.42
0.0075
-
isocitrate
pH 7.4, wild-type enzyme
Sus scrofa
1.1.1.42
0.0092
-
isocitrate
pH 7.4, mutant K212Y
Sus scrofa
1.1.1.42
0.0098
-
isocitrate
pH 7.4, mutant K212Q
Sus scrofa
1.1.1.42
0.0099
-
NADP+
pH 7.4, wild-type enzyme
Sus scrofa
1.1.1.42
0.014
-
NADP+
pH 7.4, mutant K212Q
Sus scrofa
1.1.1.42
0.018
-
NADP+
pH 7.4, mutant K212R
Sus scrofa
1.1.1.42
0.116
-
isocitrate
pH 7.4, mutant K212R
Sus scrofa
1.1.1.42
0.131
-
Mn2+
pH 7.4, mutant Y140K
Sus scrofa
1.1.1.42
0.252
-
Mn2+
pH 7.4, mutant K212R
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
43000
-
2 * 43000, recombinant wild-type and mutant enzymes, SDS-PAGE
Sus scrofa
1.1.1.42
92000
-
recombinant wild-type and mutant enzymes, native PAGE
Sus scrofa
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
1.1.1.42
Sus scrofa
P33198
-
-
Purification (Commentary)
EC Number
Commentary
Organism
1.1.1.42
recombinant wild-type and mutant enzymes from Escherichia coli, to homogeneity
Sus scrofa
Reaction
EC Number
Reaction
Commentary
Organism
1.1.1.42
isocitrate + NADP+ = 2-oxoglutarate + CO2 + NADPH + H+
Tyr140 and Lys212 are required for catalytic activity, Tyr140 is the general acid that protonates the substrate after decarboxylation, Lys212 lowers as a positively charged residue the pK of the nearby ionizable group in the enzyme-substrate complex and stabilizes the carbanion formed initially on substrate decarboxylation
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 [Ámol/min/mg]
Specific Activity Maximum [Ámol/min/mg]
Commentary
Organism
1.1.1.42
0.003
-
purified recombinant mutant K212Y, forward reaction
Sus scrofa
1.1.1.42
0.065
-
purified recombinant mutant K212Q, forward reaction
Sus scrofa
1.1.1.42
0.08
-
purified recombinant mutant Y140E, forward reaction
Sus scrofa
1.1.1.42
0.083
-
purified recombinant mutant Y140T, forward reaction
Sus scrofa
1.1.1.42
0.102
-
purified recombinant mutant Y140F, forward reaction
Sus scrofa
1.1.1.42
0.179
-
purified recombinant mutant Y140K, forward reaction
Sus scrofa
1.1.1.42
3.16
-
purified recombinant mutant K212R, forward reaction
Sus scrofa
1.1.1.42
35.1
-
purified recombinant wild-type enzyme, forward reaction
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+
-
656180
Sus scrofa
2-oxoglutarate + CO2 + NADPH
-
-
-
r
Subunits
EC Number
Subunits
Commentary
Organism
1.1.1.42
dimer
2 * 43000, recombinant 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
-
forward reaction, assay at
Sus scrofa
1.1.1.42
37
-
reverse reaction, 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
additional information
-
pH-profile, recombinant wild-type and mutant enzymes
Sus scrofa
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
1.1.1.42
NADP+
-
Sus scrofa
1.1.1.42
NADPH
-
Sus scrofa
Activating Compound (protein specific)
EC Number
Activating Compound
Commentary
Organism
Structure
1.1.1.42
acetic acid
mutant Y140T, 106fold activation compared to the wild-type enzyme
Sus scrofa
1.1.1.42
ethylamine
mutant K212Q, 4fold activation compared to the wild-type enzyme
Sus scrofa
1.1.1.42
phenol
mutant Y140T, 88fold activation compared to the wild-type enzyme
Sus scrofa
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
1.1.1.42
expression of wild-type and mutant enzymes in Escherichia coli
Sus scrofa
Cofactor (protein specific)
EC Number
Cofactor
Commentary
Organism
Structure
1.1.1.42
NADP+
-
Sus scrofa
1.1.1.42
NADPH
-
Sus scrofa
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
1.1.1.42
K212Q
site-directed mutagenesis, highly decreased activity in both reaction directions compared to the wild-type enzyme, altered pH-dependency of the activity
Sus scrofa
1.1.1.42
K212R
site-directed mutagenesis, highly decreased activity in both reaction directions compared to the wild-type enzyme, altered pH-dependency of the activity
Sus scrofa
1.1.1.42
K212Y
site-directed mutagenesis, highly decreased activity in both reaction directions compared to the wild-type enzyme, altered pH-dependency of the activity
Sus scrofa
1.1.1.42
Y140E
site-directed mutagenesis, highly decreased activity in both reaction directions compared to the wild-type enzyme, unaltered Km for isocitrate and NADP+
Sus scrofa
1.1.1.42
Y140F
site-directed mutagenesis, highly decreased activity in both reaction directions compared to the wild-type enzyme, unaltered Km for isocitrate and NADP+
Sus scrofa
1.1.1.42
Y140K
site-directed mutagenesis, highly decreased activity in both reaction directions compared to the wild-type enzyme, unaltered Km for isocitrate and NADP+
Sus scrofa
1.1.1.42
Y140T
site-directed mutagenesis, highly decreased activity in both reaction directions compared to the wild-type enzyme, unaltered Km for isocitrate and NADP+, highly increased activation by added exogenous acetic acid and phenol 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
0.00004
-
Mn2+
pH 7.4, mutant Y140F
Sus scrofa
1.1.1.42
0.000084
-
Mn2+
pH 7.4, mutant Y140E
Sus scrofa
1.1.1.42
0.00011
-
Mn2+
pH 7.4, wild-type enzyme
Sus scrofa
1.1.1.42
0.00042
-
NADP+
pH 7.4, mutant Y140E
Sus scrofa
1.1.1.42
0.00046
-
NADP+
pH 7.4, mutant Y140T
Sus scrofa
1.1.1.42
0.00051
-
NADP+
pH 7.4, mutant Y140K
Sus scrofa
1.1.1.42
0.0006
-
isocitrate
pH 7.4, mutant Y140T
Sus scrofa
1.1.1.42
0.0007
-
isocitrate
pH 7.4, mutant Y140E
Sus scrofa
1.1.1.42
0.00091
-
Mn2+
pH 7.4, mutant K212Q
Sus scrofa
1.1.1.42
0.0011
-
Mn2+
pH 7.4, mutant K212Y
Sus scrofa
1.1.1.42
0.0023
-
NADP+
pH 7.4, mutant Y140F
Sus scrofa
1.1.1.42
0.0052
-
isocitrate
pH 7.4, mutant Y140F
Sus scrofa
1.1.1.42
0.0053
-
isocitrate
pH 7.4, mutant Y140K
Sus scrofa
1.1.1.42
0.0073
-
NADP+
pH 7.4, mutant K212Y
Sus scrofa
1.1.1.42
0.0075
-
isocitrate
pH 7.4, wild-type enzyme
Sus scrofa
1.1.1.42
0.0092
-
isocitrate
pH 7.4, mutant K212Y
Sus scrofa
1.1.1.42
0.0098
-
isocitrate
pH 7.4, mutant K212Q
Sus scrofa
1.1.1.42
0.0099
-
NADP+
pH 7.4, wild-type enzyme
Sus scrofa
1.1.1.42
0.014
-
NADP+
pH 7.4, mutant K212Q
Sus scrofa
1.1.1.42
0.018
-
NADP+
pH 7.4, mutant K212R
Sus scrofa
1.1.1.42
0.116
-
isocitrate
pH 7.4, mutant K212R
Sus scrofa
1.1.1.42
0.131
-
Mn2+
pH 7.4, mutant Y140K
Sus scrofa
1.1.1.42
0.252
-
Mn2+
pH 7.4, mutant K212R
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
43000
-
2 * 43000, recombinant wild-type and mutant enzymes, SDS-PAGE
Sus scrofa
1.1.1.42
92000
-
recombinant wild-type and mutant enzymes, native PAGE
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
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 [Ámol/min/mg]
Specific Activity Maximum [Ámol/min/mg]
Commentary
Organism
1.1.1.42
0.003
-
purified recombinant mutant K212Y, forward reaction
Sus scrofa
1.1.1.42
0.065
-
purified recombinant mutant K212Q, forward reaction
Sus scrofa
1.1.1.42
0.08
-
purified recombinant mutant Y140E, forward reaction
Sus scrofa
1.1.1.42
0.083
-
purified recombinant mutant Y140T, forward reaction
Sus scrofa
1.1.1.42
0.102
-
purified recombinant mutant Y140F, forward reaction
Sus scrofa
1.1.1.42
0.179
-
purified recombinant mutant Y140K, forward reaction
Sus scrofa
1.1.1.42
3.16
-
purified recombinant mutant K212R, forward reaction
Sus scrofa
1.1.1.42
35.1
-
purified recombinant wild-type enzyme, forward reaction
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+
-
656180
Sus scrofa
2-oxoglutarate + CO2 + NADPH
-
-
-
r
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
1.1.1.42
dimer
2 * 43000, recombinant 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
-
forward reaction, assay at
Sus scrofa
1.1.1.42
37
-
reverse reaction, 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
additional information
-
pH-profile, recombinant wild-type and mutant enzymes
Sus scrofa