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 ACCESSION NO.
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 ACCESSION NO.
COMMENTARY
LITERATURE
hydrolysis of peptide bond
-
-
-
-
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
EC 3.4.99.44
-
-
formerly
-
Escherichia coli protease III
-
-
-
-
Pitrilysin
-
-
-
-
ppBH4
Bacillus halodurans H4
-
-
-
Protease Pi
-
-
-
-
Proteinase Pi
-
-
-
-
Proteinase, Escherichia coli metallo-, Pi
-
-
-
-
PTR
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY
81611-78-1
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
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 ACCESSION NO.
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 ACCESSION NO.
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 ACCESSION NO.
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
-
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
Tetraethylenepentamine
-
-
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]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
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]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
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]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
additional information
-
-
-
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
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
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
pI VALUE
pI VALUE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
5.4
-
-
calculated
LOCALIZATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
Escherichia coli (strain K12)
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
46500
-
-
gel filtration
107700
-
-
E. coli, calculation from amino acid sequence
108000
-
-
Acinetobacter calcoaceticus, gel filtration
112000
-
-
Acinetobacter calcoaceticus, native electrophoresis
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
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
TEMPERATURE STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
40
-
-
10 min, stable
50
-
-
half-life is 20 min
55
-
-
half-life is 10 min
STORAGE STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
-20C, 50 mM Tris-HCl, pH 7.5, 40% v/v glycerol
-
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
recombinant protein with His-tag
-
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
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