BRENDA - Enzyme Database

Identification of cofactor discrimination sites in NAD-isocitrate dehydrogenase from Pyrococcus furiosus

Steen, I.H.; Lien, T.; Madsen, M.S.; Birkeland, N.K.; Arch. Microbiol. 178, 297-300 (2002)

Data extracted from this reference:

Engineering
EC Number
Amino acid exchange
Commentary
Organism
1.1.1.41
D328K
the mutant shows dual coenzyme specificity
Pyrococcus furiosus
1.1.1.41
D328K/I329Y
introduction of the double mutation shifts the cofactor preference from NAD+ to NADP+
Pyrococcus furiosus
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.1.1.41
0.066
-
NAD+
pH 8.0, 70°C, mutant enzyme D328K
Pyrococcus furiosus
1.1.1.41
0.068
-
NAD+
pH 8.0, 70°C, wild-type enzyme
Pyrococcus furiosus
1.1.1.41
0.162
-
NADP+
pH 8.0, 70°C, mutant enzyme D328K
Pyrococcus furiosus
1.1.1.41
0.211
-
NADP+
pH 8.0, 70°C, mutant enzyme D328K/I329Y
Pyrococcus furiosus
1.1.1.41
0.336
-
NAD+
pH 8.0, 70°C, mutant enzyme D328K/I329Y
Pyrococcus furiosus
1.1.1.41
4.44
-
NADP+
pH 8.0, 70°C, wild-type enzyme
Pyrococcus furiosus
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
1.1.1.41
Pyrococcus furiosus
Q8U488
-
-
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.1.1.41
isocitrate + NAD+
wild-type enzyme is specific for NAD+, low activity with NADP+ as cofactor, mutant D328K shows dual coenzyme specificity, cofactor preference of mutant D328K/I329Y is shifted from NAD+ to NADP+
721450
Pyrococcus furiosus
2-oxoglutarate + CO2 + NADH
-
-
-
?
1.1.1.41
isocitrate + NADP+
wild-type enzyme is specific for NAD+, low activity with NADP+ as cofactor, mutant D328K shows dual coenzyme specificity, cofactor preference of mutant D328K/I329Y is shifted from NAD+ to NADP+
721450
Pyrococcus furiosus
2-oxoglutarate + CO2 + NADPH
-
-
-
?
Temperature Optimum [°C]
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
1.1.1.41
70
-
assay at
Pyrococcus furiosus
Temperature Range [°C]
EC Number
Temperature Minimum [°C]
Temperature Maximum [°C]
Commentary
Organism
1.1.1.41
80
95
activity at 80°C is about 50% compared to the activity at 90°C
Pyrococcus furiosus
Temperature Stability [°C]
EC Number
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
Organism
1.1.1.41
102
-
the apparent melting temperature is 102.3°C, mutant enzyme D328K
Pyrococcus furiosus
1.1.1.41
103
-
the apparent melting temperature is 102.9°C, mutant enzyme D328K/I329Y
Pyrococcus furiosus
1.1.1.41
104
-
the apparent melting temperature is 103.7°C, wild-type enzyme
Pyrococcus furiosus
Turnover Number [1/s]
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
1.1.1.41
60.4
-
NAD+
pH 8.0, 70°C, wild-type enzyme
Pyrococcus furiosus
1.1.1.41
93
-
NADP+
pH 8.0, 70°C, wild-type enzyme
Pyrococcus furiosus
1.1.1.41
103.4
-
NAD+
pH 8.0, 70°C, mutant enzyme D328K
Pyrococcus furiosus
1.1.1.41
105.6
-
NAD+
pH 8.0, 70°C, mutant enzyme D328K/I329Y
Pyrococcus furiosus
1.1.1.41
133
-
NADP+
pH 8.0, 70°C, mutant enzyme D328K
Pyrococcus furiosus
1.1.1.41
168
-
NADP+
pH 8.0, 70°C, mutant enzyme D328K/I329Y
Pyrococcus furiosus
pH Optimum
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.1.1.41
8
-
assay at
Pyrococcus furiosus
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
1.1.1.41
NAD+
wild-type enzyme is specific for NAD+, low activity with NADP+ as cofactor, mutant D328K shows dual coenzyme specificity, cofactor preference of mutant D328K/I329Y is shifted from NAD+ to NADP+
Pyrococcus furiosus
1.1.1.41
NADP+
wild-type enzyme is specific for NAD+, low activity with NADP+ as cofactor, mutant D328K shows dual coenzyme specificity, cofactor preference of mutant D328K/I329Y is shifted from NAD+ to NADP+
Pyrococcus furiosus
Cofactor (protein specific)
EC Number
Cofactor
Commentary
Organism
Structure
1.1.1.41
NAD+
wild-type enzyme is specific for NAD+, low activity with NADP+ as cofactor, mutant D328K shows dual coenzyme specificity, cofactor preference of mutant D328K/I329Y is shifted from NAD+ to NADP+
Pyrococcus furiosus
1.1.1.41
NADP+
wild-type enzyme is specific for NAD+, low activity with NADP+ as cofactor, mutant D328K shows dual coenzyme specificity, cofactor preference of mutant D328K/I329Y is shifted from NAD+ to NADP+
Pyrococcus furiosus
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
1.1.1.41
D328K
the mutant shows dual coenzyme specificity
Pyrococcus furiosus
1.1.1.41
D328K/I329Y
introduction of the double mutation shifts the cofactor preference from NAD+ to NADP+
Pyrococcus furiosus
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.1.1.41
0.066
-
NAD+
pH 8.0, 70°C, mutant enzyme D328K
Pyrococcus furiosus
1.1.1.41
0.068
-
NAD+
pH 8.0, 70°C, wild-type enzyme
Pyrococcus furiosus
1.1.1.41
0.162
-
NADP+
pH 8.0, 70°C, mutant enzyme D328K
Pyrococcus furiosus
1.1.1.41
0.211
-
NADP+
pH 8.0, 70°C, mutant enzyme D328K/I329Y
Pyrococcus furiosus
1.1.1.41
0.336
-
NAD+
pH 8.0, 70°C, mutant enzyme D328K/I329Y
Pyrococcus furiosus
1.1.1.41
4.44
-
NADP+
pH 8.0, 70°C, wild-type enzyme
Pyrococcus furiosus
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
isocitrate + NAD+
wild-type enzyme is specific for NAD+, low activity with NADP+ as cofactor, mutant D328K shows dual coenzyme specificity, cofactor preference of mutant D328K/I329Y is shifted from NAD+ to NADP+
721450
Pyrococcus furiosus
2-oxoglutarate + CO2 + NADH
-
-
-
?
1.1.1.41
isocitrate + NADP+
wild-type enzyme is specific for NAD+, low activity with NADP+ as cofactor, mutant D328K shows dual coenzyme specificity, cofactor preference of mutant D328K/I329Y is shifted from NAD+ to NADP+
721450
Pyrococcus furiosus
2-oxoglutarate + CO2 + NADPH
-
-
-
?
Temperature Optimum [°C] (protein specific)
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
1.1.1.41
70
-
assay at
Pyrococcus furiosus
Temperature Range [°C] (protein specific)
EC Number
Temperature Minimum [°C]
Temperature Maximum [°C]
Commentary
Organism
1.1.1.41
80
95
activity at 80°C is about 50% compared to the activity at 90°C
Pyrococcus furiosus
Temperature Stability [°C] (protein specific)
EC Number
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
Organism
1.1.1.41
102
-
the apparent melting temperature is 102.3°C, mutant enzyme D328K
Pyrococcus furiosus
1.1.1.41
103
-
the apparent melting temperature is 102.9°C, mutant enzyme D328K/I329Y
Pyrococcus furiosus
1.1.1.41
104
-
the apparent melting temperature is 103.7°C, wild-type enzyme
Pyrococcus furiosus
Turnover Number [1/s] (protein specific)
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
1.1.1.41
60.4
-
NAD+
pH 8.0, 70°C, wild-type enzyme
Pyrococcus furiosus
1.1.1.41
93
-
NADP+
pH 8.0, 70°C, wild-type enzyme
Pyrococcus furiosus
1.1.1.41
103.4
-
NAD+
pH 8.0, 70°C, mutant enzyme D328K
Pyrococcus furiosus
1.1.1.41
105.6
-
NAD+
pH 8.0, 70°C, mutant enzyme D328K/I329Y
Pyrococcus furiosus
1.1.1.41
133
-
NADP+
pH 8.0, 70°C, mutant enzyme D328K
Pyrococcus furiosus
1.1.1.41
168
-
NADP+
pH 8.0, 70°C, mutant enzyme D328K/I329Y
Pyrococcus furiosus
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.1.1.41
8
-
assay at
Pyrococcus furiosus
KCat/KM [mM/s]
EC Number
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
1.1.1.41
20.9
-
NADP+
pH 8.0, 70°C, wild-type enzyme
Pyrococcus furiosus
1.1.1.41
314
-
NAD+
pH 8.0, 70°C, mutant enzyme D328K/I329Y
Pyrococcus furiosus
1.1.1.41
796
-
NADP+
pH 8.0, 70°C, mutant enzyme D328K/I329Y
Pyrococcus furiosus
1.1.1.41
821
-
NADP+
pH 8.0, 70°C, mutant enzyme D328K
Pyrococcus furiosus
1.1.1.41
888
-
NAD+
pH 8.0, 70°C, wild-type enzyme
Pyrococcus furiosus
1.1.1.41
1567
-
NAD+
pH 8.0, 70°C, mutant enzyme D328K
Pyrococcus furiosus
KCat/KM [mM/s] (protein specific)
EC Number
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
1.1.1.41
20.9
-
NADP+
pH 8.0, 70°C, wild-type enzyme
Pyrococcus furiosus
1.1.1.41
314
-
NAD+
pH 8.0, 70°C, mutant enzyme D328K/I329Y
Pyrococcus furiosus
1.1.1.41
796
-
NADP+
pH 8.0, 70°C, mutant enzyme D328K/I329Y
Pyrococcus furiosus
1.1.1.41
821
-
NADP+
pH 8.0, 70°C, mutant enzyme D328K
Pyrococcus furiosus
1.1.1.41
888
-
NAD+
pH 8.0, 70°C, wild-type enzyme
Pyrococcus furiosus
1.1.1.41
1567
-
NAD+
pH 8.0, 70°C, mutant enzyme D328K
Pyrococcus furiosus