Information on EC 3.4.24.55 - pitrilysin

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The expected taxonomic range for this enzyme is: Bacteria

EC NUMBER
COMMENTARY
3.4.24.55
-
RECOMMENDED NAME
GeneOntology No.
pitrilysin
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Preferential cleavage of -Tyr16-/- Leu- and -Phe25-/- Tyr-bonds of oxidized insulin B chain. Also acts on other substrates of less than 7 kDa such as insulin and glucagon
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
hydrolysis of peptide bond
-
-
-
-
SYNONYMS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Escherichia coli protease III
-
-
-
-
Pitrilysin
-
-
-
-
Protease Pi
-
-
-
-
Proteinase Pi
-
-
-
-
Proteinase, Escherichia coli metallo-, Pi
-
-
-
-
PTR
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY
81611-78-1
-
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain H4
-
-
Manually annotated by BRENDA team
Bacillus halodurans H4
strain H4
-
-
Manually annotated by BRENDA team
overexpressing strain
-
-
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(7-Methoxycoumarin-4-yl)acetyl-Nle-Ala-Val-Lys-Tyr-Leu-Asn-Ser-(2,4-dinitrophenyl)Lys-Leu-Asp-D-Lys + H2O
?
show the reaction diagram
-
-
-
-
-
amyloid beta-peptide + H2O
?
show the reaction diagram
-
-
-
-
?
amyloid beta-peptide with mutations K16A/K28A + H2O
?
show the reaction diagram
-
cleavage site is identical to that of native amyloid beta-peptide
-
-
?
Angiotensinogen(1-14) + H2O
?
show the reaction diagram
-
-
-
-
-
beta-amyloid + H2O
?
show the reaction diagram
-
18% of the activity with beta-endorphin, major cleavage site His14-Gln15
-
-
?
beta-endorphin + H2O
?
show the reaction diagram
-
cleavage at Lys19-Asn20 bond. Residues Leu14, Val15 and Leu17 and region 22-26 are responsible for recognition by enzyme
-
-
?
calcitonin + H2O
?
show the reaction diagram
-
3% of the activity with beta-endorphin
-
-
?
dynorphin + H2O
?
show the reaction diagram
Bacillus halodurans, Bacillus halodurans H4
-
-
-
-
?
dynorphin A fragment 1-13 + H2O
?
show the reaction diagram
Bacillus halodurans, Bacillus halodurans H4
-
cleavage at Phe4-Leu5, Leu5-Arg6, Arg6-Arg7, Arg7-Ile8
-
-
?
Glucagon + H2O
?
show the reaction diagram
-
-
-
-
-
Glucagon + H2O
?
show the reaction diagram
-
-
-
-
-
Glucagon + H2O
?
show the reaction diagram
-
8% of the activity with beta-endorphin
-
-
?
Insulin + H2O
Hydrolyzed insulin
show the reaction diagram
-
-
-
-
-
Insulin + H2O
Hydrolyzed insulin
show the reaction diagram
-
-
-
-
-
Insulin + H2O
Hydrolyzed insulin
show the reaction diagram
-
degradation in such a way that its receptor binding activity is rapidly lost but its precipitability in trichloroacetic acid is only slightly decreased
-
-
-
Insulin B-chain + H2O
?
show the reaction diagram
-
-
-
-
-
Insulin B-chain + H2O
?
show the reaction diagram
-
cleavage sites are Asn3-Gln4, His10-Leu11, Ala14-Leu15, Leu17-Val18, Gly23-Phe24, Phe24-Phe25, Phe25-Tyr26, the enzyme shares one of the only two cleavage sites with pitrilysin and 4 sites with insulin-destroying insulinase (EC 3.4.24.56)
-
-
-
Insulin B-chain + H2O
?
show the reaction diagram
-
small peptides down to 10 residues in length are cleaved more slowly, intact insulin is cleaved very slowly
-
-
-
Insulin B-chain + H2O
?
show the reaction diagram
-
17% of the activity with beta-endorphin, major cleavage site Tyr16-Leu17
-
-
?
Insulin B-chain + H2O
?
show the reaction diagram
Bacillus halodurans, Bacillus halodurans H4
-
cleavage at Phe25-Tyr26, Tyr16-Leu17, His10-Leu11
-
-
?
Secretin + H2O
?
show the reaction diagram
-
-
-
-
-
Substance P + H2O
?
show the reaction diagram
-
10% of the activity with beta-endorphin
-
-
?
Thyrocalcitonin + H2O
?
show the reaction diagram
-
-
-
-
-
Vasoactive intestinal peptide + H2O
?
show the reaction diagram
-
7.2% of the activity with beta-endorphin, major cleavage site Arg14-Lys15
-
-
?
Insulin-like growth factor II + H2O
?
show the reaction diagram
-
-
-
-
-
additional information
?
-
-
-
-
-
-
additional information
?
-
-
larger proteins are accepted as substrates to a low extent
-
-
-
additional information
?
-
-
prefers the cleavage of small polypeptides to the cleavage of proteins, inactive against synthetic amino acid derivatives of subtilisin, thermolysin, trypsin and chymotrypsin
-
-
-
additional information
?
-
-
the activity of the enzyme is confined to substrates smaller than proteins
-
-
-
additional information
?
-
Bacillus halodurans, Bacillus halodurans H4
-
no substrate: 4-nitroanilides of several amino acids tested, azocasein, bovine serum albumin
-
-
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Ca2+
-
reactivation after inhibition with chelating agents
Co2+
-
reactivation after inhibition with chelating agents
Co2+
-
reactivation after inhibition with chelating agents
Mn2+
-
reactivation after inhibition with chelating agents
Mn2+
-
marked activation
Zn2+
-
contains 0.6 mol of zinc per mol of enzyme
Zn2+
-
contains 1.02-1.14 mol of zinc per mol of enzyme (depending on mutant)
Zn2+
-
most effective metal ion in reactivation after inhibition by chelating agent
Zn2+
-
most effective metal ion in reactivation after inhibition by chelating agent
Zn2+
-
protein contains 1 atom per molecule
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1,10-phenanthroline
-
-
1,10-phenanthroline
-
Co2+, and to a lesser extent but at lower metal ion concentration Zn2+ restores activity
1,10-phenanthroline
-
1 mM, 70% inhibition
2,6-pyridinedicarboxylic acid
-
-
2-mercaptoethanol
-
1% w/v, 95% inhibition
bacitracin
-
-
bacitracin
-
especially in the presence of zinc
Ca2+
-
1 mM, weak, reactivates after inhibition with chelating agents
Chelating agents
-
-
Co2+
-
1 mM, complete inhibition
Cu2+
-
1 mM, complete inhibition
cysteine
-
1 mM, complete inhibition
dynorphin A(1-13)
-
-
E-64
-
1 mM, 46% inhibition
EDTA
-
Co2+, and to a lesser extent but at lower metal ion concentration Zn2+ restores activity
EDTA
-
1 mM, complete inhibition
EGTA
-
1 mM, complete inhibition
Fe2+
-
1 mM, 76% inhibition
N-ethylmaleimide
-
1 mM, 95% inhibition
phosphoramidon
-
-
Tetraethylenepentamine
-
-
Zincov
-
-
Zn2+
-
1 mM inhibits, but reactivates after inhibition with chelating agents
Zn2+
-
1 mM, complete inhibition
Mn2+
-
1 mM, weak, reactivates after inhibition with chelating agents
additional information
-
not: sulfhydryl-modifying agents
-
additional information
-
alpha-macroglobulin
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0077
(7-methoxycoumarin-4-yl)acetyl-Nle-Ala-Val-Lys-Tyr-Leu-Asn-Ser-Lys(2,4-dinitrophenyl)-Leu-Asp-D-Lys
-
-
0.0009
beta-amyloid
-
pH 8.0, 37C
-
0.00036
beta-endorphin
-
pH 8.0, 37C
-
0.0022
calcitonin
-
pH 8.0, 37C
0.12
dynorphin A fragment 1-13
-
pH 8.0, 37C
0.00061
Glucagon
-
-
0.0127
Glucagon
-
-
0.000105
Insulin
-
-
-
0.0015
Insulin B-chain
-
pH 8.0, 37C
0.05
Insulin B-chain
-
pH 8.0, 37C
0.031
Substance P
-
pH 8.0, 37C
0.0004
Vasoactive intestinal peptide
-
pH 8.0, 37C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
2.9
(7-Methoxycoumarin-4-yl)acetyl-Nle-Ala-Val-Lys-Tyr-Leu-Asn-Ser-(2,4-dinitrophenyl)Lys-Leu-Asp-D-Lys
-
-
2.33
beta-amyloid
-
pH 8.0, 37C
-
12.5
beta-endorphin
-
pH 8.0, 37C
-
0.43
calcitonin
-
pH 8.0, 37C
0.55
dynorphin A fragment 1-13
-
pH 8.0, 37C
0.05
Glucagon
-
-
0.18
Insulin B-chain
-
pH 8.0, 37C
2.66
Insulin B-chain
-
pH 8.0, 37C
0.63
Substance P
-
pH 8.0, 37C
0.9
Vasoactive intestinal peptide
-
pH 8.0, 37C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
6.8 - 7.2
-
insulin
7.5
-
4-dinitrophenyl)Lys-Leu-Asp-D-Lys
8
-
activity in sodium phosphate buffer is 3- to 4fold higher than in veronal or Tris/HCl buffer
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
5.4
-
calculated
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
Escherichia coli (strain K12)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
46500
-
gel filtration
667795
107700
-
E. coli, calculation from amino acid sequence
31302
108000
-
Acinetobacter calcoaceticus, gel filtration
31310
112000
-
Acinetobacter calcoaceticus, native electrophoresis
31310
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
monomer
-
1 * 116000, SDS-PAGE
monomer
-
1 * 107000, Acinetobacter calcoaceticus, SDS-PAGE
monomer
-
1 * 46500, SDS-PAGE, 1 * 46585, calculated
monomer
Bacillus halodurans H4
-
1 * 46500, SDS-PAGE, 1 * 46585, calculated
-
tetramer
-
4 * 27000, Acinetobacter calcoaceticus, SDS-PAGE
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
40
-
10 min, stable
667795
50
-
half-life is 20 min
667795
55
-
half-life is 10 min
667795
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 50 mM Tris-HCl, pH 7.5, 40% v/v glycerol
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant protein with His-tag
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D79A
-
no effect on function
E91A
-
no proteolytic activity
F198A
-
enzyme remains functional
H193A
-
enzyme remains functional
N119A
-
promotion of yeast mating factor in heterologous expression is disabled
N317A/R318A/S319A
-
enzyme remains functional
R792A
-
no proteolytic activity
R792A/T793A/E794A/E795A
-
no proteolytic activity
S117A
-
enzyme remains functional
S117A/H118A/N119A
-
promotion of yeast mating factor in heterologous expression is disabled
S196A/K197A/F198A/S199A
-
promotion of yeast mating factor in heterologous expression is disabled
T127A
-
enzyme remains functional
T322A/L323A
-
enzyme remains functional
Y799A
-
no proteolytic activity
K197A
-
enzyme remains functional
additional information
-
heterologous expression of enzyme in yeast, enzyme functionally substitutes for yeast Ax11p and Ste23p in pheromone production. Independent heterologous expression of N-terminal domain with amino acids 1-515 or C-terminal domain with amino acids 516-962 does not promote yeast mating. Co-expression of both domains restores yeast mating