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Literature summary for 3.4.24.27 extracted from

  • Menach, E.; Yasukawa, K.; Inouye, K.
    Effects of site-directed mutagenesis of the loop residue of the N-terminal domain Gly117 of thermolysin on its catalytic activity (2010), Biosci. Biotechnol. Biochem., 74, 2457-2462.
    View publication on PubMed
Search for more literature for 3.4.24.27

Cloned(Commentary)

Cloned (Comment) Organism
expression of wild-type and mutant enzymes in Escherichia coli strain T5KC991 Bacillus thermoproteolyticus

Protein Variants

Protein Variants Comment Organism
G117D site-directed mutagenesis, the mutant enzyme shows reduced activity compared to the wild-type Bacillus thermoproteolyticus
G117E site-directed mutagenesis, the mutant enzyme shows increased activity compared to the wild-type enzyme, the kcat/Km value is 80% of wild-type level with N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide, but 130% with N-benzyloxycarbonyl-L-Asp-L-Phe-methyl ester Bacillus thermoproteolyticus
G117K site-directed mutagenesis, the mutant enzyme shows reduced activity compared to the wild-type, the kcat/Km value is 40% of wild-type level with N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide, but 80% with N-benzyloxycarbonyl-L-Asp-L-Phe-methyl ester Bacillus thermoproteolyticus
G117R site-directed mutagenesis, the mutant enzyme shows reduced activity compared to the wild-type, the kcat/Km value is 40% of wild-type level with N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and N-benzyloxycarbonyl-L-Asp-L-Phe-methyl ester Bacillus thermoproteolyticus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information Michaelis-Menten kinetics, overview Bacillus thermoproteolyticus
0.42
-
 N-benzyloxycarbonyl-L-Asp-L-Phe-methyl ester recombinant wild-type enzyme, pH 7.5, 25°C Bacillus thermoproteolyticus
0.44
-
 N-benzyloxycarbonyl-L-Asp-L-Phe-methyl ester recombinant mutant G117E, pH 7.5, 25°C Bacillus thermoproteolyticus
0.5
-
 N-benzyloxycarbonyl-L-Asp-L-Phe-methyl ester recombinant mutant G117R, pH 7.5, 25°C Bacillus thermoproteolyticus
0.57
-
 N-benzyloxycarbonyl-L-Asp-L-Phe-methyl ester recombinant mutant G117K, pH 7.5, 25°C Bacillus thermoproteolyticus

Metals/Ions

Metals/Ions Comment Organism Structure
Ca2+ required Bacillus thermoproteolyticus
NaCl induces enzyme activation, activation of mutant enzymes is reduced compared to the wild-type enzyme Bacillus thermoproteolyticus

Organism

Organism UniProt Comment Textmining
Bacillus thermoproteolyticus
-
-
-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
2000
-
 recombinant mutant G117D, substrate casein, pH 7.5, 25°C Bacillus thermoproteolyticus
2900
-
 recombinant mutant G117R, substrate casein, pH 7.5, 25°C Bacillus thermoproteolyticus
3200
-
 recombinant mutant G117K, substrate casein, pH 7.5, 25°C Bacillus thermoproteolyticus
11000
-
 recombinant wild-type enzyme, substrate casein, pH 7.5, 25°C Bacillus thermoproteolyticus
12000
-
 recombinant mutant G117E, substrate casein, pH 7.5, 25°C Bacillus thermoproteolyticus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
casein + H2O
-
 Bacillus thermoproteolyticus ?
-
 ?
N-benzyloxycarbonyl-L-Asp-L-Phe-methyl ester + H2O
-
 Bacillus thermoproteolyticus N-benzyloxycarbonyl-L-Asp + L-Phe-methyl ester
-
 ?
N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide + H2O
-
 Bacillus thermoproteolyticus N-[3-(2-furyl)acryloyl]-glycine + L-leucine amide
-
 ?

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
 assay at Bacillus thermoproteolyticus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
4.8
-
 N-benzyloxycarbonyl-L-Asp-L-Phe-methyl ester recombinant mutant G117R, pH 7.5, 25°C Bacillus thermoproteolyticus
7.9
-
 N-benzyloxycarbonyl-L-Asp-L-Phe-methyl ester recombinant wild-type enzyme, pH 7.5, 25°C Bacillus thermoproteolyticus
8.4
-
 N-benzyloxycarbonyl-L-Asp-L-Phe-methyl ester recombinant mutant G117K, pH 7.5, 25°C Bacillus thermoproteolyticus
10.5
-
 N-benzyloxycarbonyl-L-Asp-L-Phe-methyl ester recombinant mutant G117E, pH 7.5, 25°C Bacillus thermoproteolyticus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
 assay at Bacillus thermoproteolyticus

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
9.6
-
 N-benzyloxycarbonyl-L-Asp-L-Phe-methyl ester recombinant mutant G117R, pH 7.5, 25°C Bacillus thermoproteolyticus
14.7
-
 N-benzyloxycarbonyl-L-Asp-L-Phe-methyl ester recombinant mutant G117K, pH 7.5, 25°C Bacillus thermoproteolyticus
15
-
 N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide recombinant mutant G117R, pH 7.5, 25°C Bacillus thermoproteolyticus
16
-
 N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide recombinant mutant G117K, pH 7.5, 25°C Bacillus thermoproteolyticus
18.8
-
 N-benzyloxycarbonyl-L-Asp-L-Phe-methyl ester recombinant wild-type enzyme, pH 7.5, 25°C Bacillus thermoproteolyticus
23.9
-
 N-benzyloxycarbonyl-L-Asp-L-Phe-methyl ester recombinant mutant G117E, pH 7.5, 25°C Bacillus thermoproteolyticus
33
-
 N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide recombinant mutant G117E, pH 7.5, 25°C Bacillus thermoproteolyticus
40
-
 N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide recombinant wild-type enzyme, pH 7.5, 25°C Bacillus thermoproteolyticus