Information on EC 3.4.21.66 - Thermitase

Word Map on EC 3.4.21.66
Please wait a moment until all data is loaded. This message will disappear when all data is loaded.
Specify your search results
Select one or more organisms in this record:
Show additional data
Do not include text mining results
Include (text mining) results (more...)
Include results (AMENDA + additional results, but less precise; more...)

The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
3.4.21.66
-
RECOMMENDED NAME
GeneOntology No.
Thermitase
-
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Hydrolysis of proteins, including collagen
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of peptide bond
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY hide
69772-87-8
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
gene aprE
UniProt
Manually annotated by BRENDA team
isolated from forest soil in Thailand
-
-
Manually annotated by BRENDA team
Streptomyces rectus var. proteolyticus
ATCC 21067
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
proteolysin is a serine protease of the thermitase subgroup of subtilases, phylogenetic tree
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
Acetyl-(L-Ala)3 methyl ester + H2O
?
show the reaction diagram
-
best substrate
-
-
-
Anionic protease + H2O
?
show the reaction diagram
-
at pH 8 or 6 and 25°C or 4°C completely hydrolyzed
-
-
-
azocasein + H2O
?
show the reaction diagram
Benzyloxycarbonyl-L-Ala-L-Ala-L-Leu 4-nitroanilide + H2O
?
show the reaction diagram
-
-
-
-
-
Carbobenzoxy-alanine 4-nitrophenyl ester + H2O
?
show the reaction diagram
Streptomyces rectus var. proteolyticus
-
-
-
-
-
casein + H2O
?
show the reaction diagram
casein + H2O
hydrolyzed casein
show the reaction diagram
Collagen + H2O
Hydrolyzed collagen
show the reaction diagram
-
-
-
-
-
Cry11A endotoxin + H2O
?
show the reaction diagram
-
substrate from Bacillus thuringiensis ssp. israelensis, enzyme hydrolyzes the I28-A29 bond. At increasing concentration of enzyme, additionally lysis of M71-E72 bond
-
-
?
Elastin + H2O
Hydrolyzed elastin
show the reaction diagram
Field bean protein + H2O
Hydrolyzed field bean protein
show the reaction diagram
-
-
-
-
-
Gelatin + H2O
?
show the reaction diagram
Gelatin + H2O
Hydrolyzed gelatin
show the reaction diagram
-
-
-
-
-
Gluten + H2O
Hydrolyzed gluten
show the reaction diagram
-
-
-
-
-
human skin elastin + H2O
?
show the reaction diagram
-
-
-
-
?
N-Acetyl-L-Tyr ethyl ester + H2O
?
show the reaction diagram
-
-
-
-
-
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide + H2O
N-succinyl-Ala-Ala-Pro-Phe + 4-nitroaniline
show the reaction diagram
N-succinyl-L-Ala-L-Ala-L-Ala 4-nitroanilide + H2O
?
show the reaction diagram
oxidized insulin B chain + H2O
?
show the reaction diagram
peptidolytic activity, primary cleavage site of proteolysin is located between Ala14 and Leu15. Prolonged incubation of 24 h reveals additional cleavage sites after Phe1, Asn3, Gln4, Leu17, Cys19, Phe24, Phe25, Tyr25, and Lys29, which shows that proteolysin has a relaxed cleavage site specificity
-
-
?
Serum albumin + H2O
Hydrolyzed serum albumin
show the reaction diagram
-
-
-
-
-
Succinyl-Ala-Ala-Phe 4-nitroanilide + H2O
?
show the reaction diagram
-
-
-
-
-
Urea-denatured hemoglobin + H2O
Hydrolyzed urea-denatured hemoglobin
show the reaction diagram
-
-
-
-
-
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
-
activates to 119% at 1 mM
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
-
at 5 mM
2,2,5,5-Tetramethylpyrrolin-1-oxyl-3-carbonyl-Ala2-Phe methyl ketone
-
-
2,2,5,5-Tetramethylpyrrolin-1-oxyl-3-carbonyl-Ala3-Phe methyl ketone
-
-
2-mercaptoethanol
-
slight inhibition
actinomycin
-
SG3, and D
Benzyloxycarbonyl-(Ala)n-Phe methyl ketone
-
reacts irreversibly in a 1:1 ratio with the enzyme
Benzyloxycarbonyl-Ala2-Phe chloromethyl ketone
-
and the corresponding methyl ketone
Benzyloxycarbonyl-Phe chloromethyl ketone
-
-
Benzyloxycarybonyl-(L-Ala)3-L-Phe methyl ketone
-
-
Ca2+
-
18% inhibition at 1 mM, 85% inhibition at 4 mM
Chloromethyl ketones
-
irreversible
Cu2+
-
8% inhibition at 1 mM, 99% inhibition at 4 mM
Dansyl-(Ala)n-PheCH2Cl
-
n: 1, 2 and 3, and homologous carboxybenzoyl-protected peptides, with a higher inhibitory effect
DTT
-
slight inhibition at 1-10 mM
EGTA
-
strong inhibition at 1 mM
Fe2+
-
54% inhibition at 1 mM, 80% inhibition at 4 mM
guanidinium hydrochloride
6 M guanidinium hydrochloride gives a 40% reduction of activity after 1 h of incubation at 40°C
H2O2
15% v/v, 90% inhibition
Inhibitor from potatoes
-
purification of two stable inhibitors
-
K+
-
36% inhibition at 1 mM, 53% inhibition at 4 mM
Methyl ketones of N-acylated peptides
-
reversible
-
Mg2+
-
54% inhibition at 1 mM, 89% inhibition at 4 mM
Na+
-
35% inhibition at 1 mM, 54% inhibition at 4 mM
p-chloromercuribenzoate
-
-
Peptide diazomethyl ketones
-
reacts irreversibly in a 1:1 ratio with the enzyme
Peptide methyl ketones
-
kinetic studies on the binding of N-acetylated peptide ketones as substrate analogous
Plysurf A210G
-
strong inhibition
-
SDS
-
strong inhibition
Triton X-100
-
-
Tween 20
-
strong inhibition
Tween 80
-
strong inhibition
Urea
-
slight inhibition
Zn2+
-
65% inhibition at 1 mM, 81% inhibition at 4 mM
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Cetyltrimethylammonium bromide
CTAB, 5% v/v, activates to 178% activity
additional information
-
cysteine residues essential for activity
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.33 - 1.96
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
25 - 248
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
62 - 335
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide
953
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
262
-
purified native enzyme, substrate gelatin, pH 9.0, 60°C
328
-
purified native enzyme, substrate N-succinyl-L-Ala-L-Ala-L-Ala 4-nitroanilide, pH 9.0, 60°C
340
-
purified native enzyme, substrate casein, pH 9.0, 60°C
1483
-
purified recombinant enzyme, pH 8.0, 60°C
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 9
-
small differences between the individual high-MW and low-MW substrates
8.2
-
benzyloxycarbonyl-Ala-Ala-Leu 4-nitroanilide
additional information
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 11
-
activity range, recombinant enzyme
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40 - 70
substrate N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide
75
-
amino acid 4-nitroanilides
additional information
-
the temperature optimum increases with incresing MW of substrate
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 80
activity range, recombinant enzyme
50 - 80
-
activity range, recombinant enzyme
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8500
-
recombinant enzyme, gel filtration, thermitase interactions with the column matrix can delay elution of the protein and lead to underestimation of the molecular size
11000
-
Thermoactinomyces vulgaris, gel filtration
21500
Streptomyces rectus var. proteolyticus
-
Streptomyces rectus var. proteolyticus, gel filtration, sedimentation equilibrium
26200
Streptomyces rectus var. proteolyticus
-
Streptomyces rectus var. proteolyticus, sedimentation diffusion measurement
28369
-
1 * 28369, Thermoactinomyces vulgaris, calculation from primary structure
28370
-
Thermoactinomyces vulgaris, calculation from primary structure
29100
Streptomyces rectus var. proteolyticus
-
Streptomyces rectus var. proteolyticus, amino acid analysis
37400
-
Thermoactinomyces vulgaris
additional information
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
a new crystal form of native thermitase has been obtained using formate as the precipitating agent
-
at 2.2 A resolution
-
comparison of the refined three-dimensional structure
-
crystal structure at 1.4 A resolution
-
crystallographic studies at 0.5 and 100 mM calcium
-
thermitase-eglin-c complex
-
thermitase-eglin-c complex (at 1.98 A resolution, and comparison of 2 crystal forms that differ in calcium content)
-
three-dimensional structure comparison of native thermitase and thermitase-eglin-c complexes
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 11
-
purified recombinant enzyme, 50°C, 5 h, over 60% activity
732871
6 - 7.5
-
maximal stability
29563
8.5 - 10.5
-
purified native enzyme, stable at
732254
additional information
-
autolysis and thereby inactivation at alkaline pH
29549
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60
-
denaturation above
80 - 100
thermostability profiles of the purified recombinant enzyme with different substrates, overview
additional information
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
Anionic protease, a component which frequently contaminates preparations of routinely isolated cationic protease, stabilizes thermitase
-
Autolysis and thereby inactivation at elevated temperature, at alkaline pH-values and in the absence of added substrate
-
Ca2+ stabilizes against both autolysis and thermal denaturation
-
Irreversible inhibition by chloromethyl ketones causes marked stabilization against thermal denaturation, reversible inhibitors have no influence on stability
-
The stability is significantly improved by 1 M acetate and chloride
-
Unusually tight binding of Ca by thermitase emerges as the most likely single influence responsible for its increased thermostability
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
autolytic processes may take place during purification
-
native enzyme 15.3fold to homogeneity from culture supernatant by dialysis, cation exchange chromatography, and again dialysis
-
recombinant enzyme from culture supernatant by dialysis and hydrophobic interaction chromatography
-
recombinant wild-type and mutant N-terminally maltose-binding protein-tagged and C-terminally His6-tagged enzymes from Escherichia coli strain C43(DE3) to near homogeneity from crude cell lysate by heat treatment at 80°C for 3 h, and ultrafiltration
single step fine purification
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DNA and amino acid sequence determination and analysis, phylogenetic tree
-
gene aprE, DNA and amino acid sequence determination and analysis, phylogenetic tree, recombinant expression of wild-type and mutant N-terminally maltose-binding protein-tagged and C-terminally His6-tagged enzymes in Escherichia coli strain C43(DE3)
recombinant expression in Lactococcus lactis strain P170 from vector pAMJ2008 and in strain MG1363, secretion of stable thermitase in an auto-induced fermentation setup at 30°C. The enzyme accumulates in the culture supernatant during batch fermentations and is easily activated at 50°C or by prolonged dialysis
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C182A/C201A
site-directed mutagenesis, elimination of the disulfide bond, the mutant shows increased activity but reduced thermostability compared to the wild-type enzyme
additional information
computational procedure OptGraft for placing a novel binding pocket onto a protein structure so as its geometry is minimally perturbed. Transfer of a calcium-binding pocket from thermitase protein, PDB entry 1thm, into the first domain of rat CD2 protein, PDB entry 1hng results in new proteins that all exhibit high affinities for terbium and can selectively bind calcium over magnesium
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
-
use of enzyme for generation of peptides for mass spectrometric characterization
biotechnology
computational procedure OptGraft for placing a novel binding pocket onto a protein structure so as its geometry is minimally perturbed. Transfer of a calcium-binding pocket from thermitase protein, PDB entry 1thm, into the first domain of rat CD2 protein, PDB entry 1hng results in new proteins that all exhibit high affinities for terbium and can selectively bind calcium over magnesium
Show AA Sequence (190 entries)
Please use the Sequence Search for a specific query.