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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 Asn116 in the beta-hairpin of the N-terminal domain of thermolysin on its activity and stability (2012), J. Biochem., 152, 231-239.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
recombinant overexpression of wild-type and mutant enzymes in Escherichia coli Bacillus thermoproteolyticus

Protein Variants

Protein Variants Comment Organism
N116A site-directed mutagenesis, the mutant shows slightly decreased activity compared to the wild-type enzyme Bacillus thermoproteolyticus
N116D site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Bacillus thermoproteolyticus
N116Q site-directed mutagenesis, the mutant shows unaltered activity compared to the wild-type enzyme Bacillus thermoproteolyticus
N116T site-directed mutagenesis, the mutant shows slightly decreased activity compared to the wild-type enzyme Bacillus thermoproteolyticus

General Stability

General Stability Organism
Ca2+ stablizes the enzyme Bacillus thermoproteolyticus
important role of Asn116 in the activity and stability of thermolysin presumably by stabilizing the beta-hairpin structure. In the N-terminal domain of thermolysin, two antiparallel beta-strands, Asn112-Ala113-Phe114-Trp115 and Ser118-Gln119-Met120-Val121-Tyr122 are connected by an Asn116-Gly117 turn to form a beta-hairpin structure Bacillus thermoproteolyticus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information Michaelis-Menten kinetics Bacillus thermoproteolyticus
0.38
-
N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester purified recombinant mutant N116Q, pH 7.5, 25°C Bacillus thermoproteolyticus
0.39
-
N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester purified recombinant wild-type enzyme, pH 7.5, 25°C Bacillus thermoproteolyticus
0.41
-
N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester purified recombinant mutant N116A, pH 7.5, 25°C Bacillus thermoproteolyticus
0.48
-
N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester purified recombinant mutant N116D, pH 7.5, 25°C Bacillus thermoproteolyticus
0.49
-
N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester purified recombinant mutant N116T, pH 7.5, 25°C Bacillus thermoproteolyticus

Metals/Ions

Metals/Ions Comment Organism Structure
Ca2+ stablizes the enzyme Bacillus thermoproteolyticus

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
34600
-
x * 34600, recombinant mature enzyme, SDS-PAGE Bacillus thermoproteolyticus

Organism

Organism UniProt Comment Textmining
Bacillus thermoproteolyticus P00800
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant mature wild-type and mutant enzymes 7.2-11fold from Escherichia coli by hydrophobic interaction and affinity chromatography Bacillus thermoproteolyticus

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
additional information
-
one proteolytic unit is defined as the amount which liberates a quantity of acid-soluble peptides that corresponds to an increase in A275 of 0.0074 (A275 of 1 mg of tyrosine)/min Bacillus thermoproteolyticus
5300
-
purified recombinant mutant N116A, pH 7.5, 25°C, substrate N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide Bacillus thermoproteolyticus
5400
-
purified recombinant mutant N116T, pH 7.5, 25°C, substrate N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide Bacillus thermoproteolyticus
6800
-
purified recombinant mutant N116Q, pH 7.5, 25°C, substrate N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide Bacillus thermoproteolyticus
7900
-
purified recombinant mutant N116D, pH 7.5, 25°C, substrate N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide Bacillus thermoproteolyticus
12000
-
purified recombinant wild-type enzyme, pH 7.5, 25°C, substrate N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide Bacillus thermoproteolyticus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
casein + H2O from bovine milk Bacillus thermoproteolyticus L-tyrosine + ?
-
?
N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester + H2O
-
Bacillus thermoproteolyticus ?
-
?
N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide + H2O
-
Bacillus thermoproteolyticus ?
-
?

Subunits

Subunits Comment Organism
? x * 34600, recombinant mature enzyme, SDS-PAGE Bacillus thermoproteolyticus

Temperature Optimum [°C]

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

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
80
-
first-order rate constant of the thermal inactivation at 80°C in the presence of 1-100mM CaCl2 Bacillus thermoproteolyticus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
5.4
-
N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester purified recombinant mutant N116T, pH 7.5, 25°C Bacillus thermoproteolyticus
6.7
-
N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester purified recombinant mutant N116A, pH 7.5, 25°C Bacillus thermoproteolyticus
7.1
-
N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester purified recombinant mutant N116Q, pH 7.5, 25°C Bacillus thermoproteolyticus
7.7
-
N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester purified recombinant wild-type enzyme, pH 7.5, 25°C Bacillus thermoproteolyticus
12
-
N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester purified recombinant mutant N116D, pH 7.5, 25°C Bacillus thermoproteolyticus

pH Optimum

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

pH Range

pH Minimum pH Maximum Comment Organism
5 8.5 activity range, profile overview Bacillus thermoproteolyticus

General Information

General Information Comment Organism
additional information structure analsis using enzyme structure PDB ID 8TLN. Catalytically important residues Glu143 and His231. Important role of Asn116 in the activity and stability of thermolysin presumably by stabilizing the beta-hairpin structure. In the N-terminal domain of thermolysin, two antiparallel beta-strands, Asn112-Ala113-Phe114-Trp115 and Ser118-Gln119-Met120-Val121-Tyr122 are connected by an Asn116-Gly117 turn to form a beta-hairpin structure Bacillus thermoproteolyticus

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
11
-
N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester purified recombinant mutant N116T, pH 7.5, 25°C Bacillus thermoproteolyticus
16
-
N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester purified recombinant mutant N116A, pH 7.5, 25°C Bacillus thermoproteolyticus
19
-
N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester purified recombinant mutant N116Q, pH 7.5, 25°C Bacillus thermoproteolyticus
20
-
N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester purified recombinant wild-type enzyme, pH 7.5, 25°C Bacillus thermoproteolyticus
26
-
N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide purified recombinant mutant N116T, pH 7.5, 25°C Bacillus thermoproteolyticus
27
-
N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester purified recombinant mutant N116D, pH 7.5, 25°C Bacillus thermoproteolyticus
31
-
N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide purified recombinant mutant N116A, pH 7.5, 25°C Bacillus thermoproteolyticus
37
-
N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide purified recombinant mutant N116Q, pH 7.5, 25°C Bacillus thermoproteolyticus
37
-
N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide purified recombinant wild-type enzyme, pH 7.5, 25°C Bacillus thermoproteolyticus
117
-
N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide purified recombinant mutant N116D, pH 7.5, 25°C Bacillus thermoproteolyticus