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Literature summary for 1.8.1.4 extracted from

  • Jolley, K.A.; Russell, R.J.; Hough, D.W.; Danson, M.J.
    Site-directed mutagenesis and halophilicity of dihydrolipoamide dehydrogenase from the halophilic archaeon, Haloferax volcanii (1997), Eur. J. Biochem., 248, 362-368.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
expression in a strain of Haloferax volcanii lacking dihydrolipoamide dehydrogenase activity Haloferax volcanii

Protein Variants

Protein Variants Comment Organism
E423A in 2 M KCl, the mutant is significantly less active than wild-type, decreased Km value for dihydrolipoamide. The mutant enzyme is not significantly activated by high salt concentrations. In the presence of 2 M KCI the thermal stability of the mutant enzyme is slightly higher than that of the wild-type enzyme Haloferax volcanii
E423D wild-type and E423D mutant enzyme are much less active in the absence of KCl than in its presence. The mutant enzyme is inactivated at temperatures around 20°C lower than the wild-type Haloferax volcanii
E423Q in 2 M KCl, the mutant is significantly less active than wild-type, whereas Km, differences are not significant. The mutant enzyme is not significantly activated by high salt concentrations. In the presence of 2 M KCI the thermal stability of the mutant enzyme is slightly higher than that of the wild-type enzyme Haloferax volcanii
E423S in 2 M KCl, the mutant is significantly less active than wild-type, whereas Km, differences are not significant. The mutant enzyme is not significantly activated by high salt concentrations. In the presence of 2 M KCI the thermal stability of the mutant enzyme is slightly higher than that of the wild-type enzyme Haloferax volcanii

Inhibitors

Inhibitors Comment Organism Structure
dihydrolipoamide
-
Haloferax volcanii

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0107
-
dihydrolipoamide pH 7.0, 30°C, mutant enzyme E423A Haloferax volcanii
0.0157
-
dihydrolipoamide pH 7.0, 30°C, mutant enzyme E423D, assay in the absence of KCI Haloferax volcanii
0.0177
-
dihydrolipoamide pH 7.0, 30°C, mutant enzyme E423Q, assay in the absence of KCI Haloferax volcanii
0.0228
-
dihydrolipoamide pH 7.0, 30°C, mutant enzyme E423Q Haloferax volcanii
0.023
-
dihydrolipoamide pH 7.0, 30°C, mutant enzyme E423S Haloferax volcanii
0.0243
-
dihydrolipoamide pH 7.0, 30°C, mutant enzyme E423A, assay in the absence of KCI Haloferax volcanii
0.0266
-
dihydrolipoamide pH 7.0, 30°C, mutant enzyme E423D Haloferax volcanii
0.0269
-
dihydrolipoamide pH 7.0, 30°C, wild-type enzyme Haloferax volcanii
0.0274
-
dihydrolipoamide pH 7.0, 30°C, wild-type enzyme, assay in the absence of KCI Haloferax volcanii
0.0364
-
dihydrolipoamide pH 7.0, 30°C, mutant enzyme E423S, assay in the absence of KCI Haloferax volcanii
0.156
-
NAD+ pH 7.0, 30°C, wild-type enzyme Haloferax volcanii
0.182
-
NAD+ pH 7.0, 30°C, mutant enzyme E423A, assay in the absence of KCI Haloferax volcanii
0.186
-
NAD+ pH 7.0, 30°C, wild-type enzyme, assay in the absence of KCI Haloferax volcanii
0.199
-
NAD+ pH 7.0, 30°C, mutant enzyme E423D Haloferax volcanii
0.203
-
NAD+ pH 7.0, 30°C, mutant enzyme E423Q Haloferax volcanii
0.211
-
NAD+ pH 7.0, 30°C, mutant enzyme E423S Haloferax volcanii
0.215
-
NAD+ pH 7.0, 30°C, mutant enzyme E423A Haloferax volcanii
0.288
-
NAD+ pH 7.0, 30°C, mutant enzyme E423Q, assay in the absence of KCI Haloferax volcanii
0.292
-
NAD+ pH 7.0, 30°C, mutant enzyme E423D, assay in the absence of KCI Haloferax volcanii
0.312
-
NAD+ pH 7.0, 30°C, mutant enzyme E423S, assay in the absence of KCI Haloferax volcanii

Organism

Organism UniProt Comment Textmining
Haloferax volcanii Q04829
-
-
Haloferax volcanii DSM 3757 Q04829
-
-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
3.6
-
pH 7.0, 30°C, mutant enzyme E423Q, assay in the absence of KCI Haloferax volcanii
4.6
-
pH 7.0, 30°C, mutant enzyme E423A, assay in the absence of KCI Haloferax volcanii
4.9
-
pH 7.0, 30°C, mutant enzyme E423D, assay in the absence of KCI Haloferax volcanii
5
-
pH 7.0, 30°C, mutant enzyme E423S, assay in the absence of KCI Haloferax volcanii
5.6
-
pH 7.0, 30°C, wild-type enzyme, assay in the absence of KCI Haloferax volcanii
5.8
-
pH 7.0, 30°C, mutant enzyme E423S, assay in the presence of 2 M KCI Haloferax volcanii
6.2
-
pH 7.0, 30°C, mutant enzyme E423A, assay in the presence of 2 M KCI Haloferax volcanii
6.4
-
pH 7.0, 30°C, mutant enzyme E423Q, assay in the presence of 2 M KCI Haloferax volcanii
16.2
-
pH 7.0, 30°C, mutant enzyme E423D, assay in the presence of 2 M KCI Haloferax volcanii
19.1
-
pH 7.0, 30°C, wild-type enzyme, assay in the presence of 2 M KCI Haloferax volcanii

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
dihydrolipoamide + NAD+
-
Haloferax volcanii lipoamide + NADH
-
?
dihydrolipoamide + NAD+
-
Haloferax volcanii DSM 3757 lipoamide + NADH
-
?

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
assay at Haloferax volcanii

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
additional information
-
in thepresence of 2 M KCI, the thermal stability properties of mutant enzymes E423S, E423Q and E423A are comparable to those of wild-type, although the mutants appear to be slightly more stable. Mutant enzyme E423D however, is inactivated at temperatures around 20°C lower than for the wild-type. In the absence of salt, similar relative thermostabilities are observed, but both wild-type and mutant enzymes are inactivated at temperatures around 16-18°C lower than in the presence of salt Haloferax volcanii

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7
-
assay at Haloferax volcanii
8.5
-
wild-type enzyme and mutant enzymes E423D, E423Q, E423S, and E423A Haloferax volcanii

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
0.023
-
dihydrolipoamide pH 7.0, 30°C Haloferax volcanii