Information on EC 3.4.24.72 - fibrolase

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY hide
3.4.24.72
-
RECOMMENDED NAME
GeneOntology No.
fibrolase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Hydrolysis of -Ala14-/-Leu- in insulin B chain and -Lys413-/-Leu- in Aalpha-chain of fibrinogen
show the reaction diagram
hydrolysis of Ala14-Leu15
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of peptide bond
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY hide
116036-70-5
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Agkistrodon halys brevicaudus
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
4-amino-benzene-His-Thr-Glu-Lys-leu-Val-Thr-Ser-dinitrophenol + H2O
?
show the reaction diagram
-
-
-
-
?
Abz-His-Thr-Glu-Lys-Leu-Val-Thr-Ser-2,4-dinitrophenyl + H2O
Abz-His-Thr-Glu-Lys + Leu-Val-Thr-Ser-2,4-dinitrophenyl
show the reaction diagram
-
-
-
-
?
alpha2-macroglobulin + H2O
fragments of alpha2-macroglobulin
show the reaction diagram
-
-
-
-
?
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg + H2O
Arg-Pro-Pro-Gly-Phe-Ser-Pro + Phe-Arg
show the reaction diagram
-
-
-
-
ir
azocasein + H2O
?
show the reaction diagram
bovine albumin + H2O
?
show the reaction diagram
-
-
-
-
?
bovine gamma-globulin + H2O
?
show the reaction diagram
-
-
-
-
?
bovine plasma fibrinogen + H2O
?
show the reaction diagram
bovine serum gamma-globulin + H2O
?
show the reaction diagram
-
marginal hydrolytic activity
-
-
?
bradykinin + H2O
Arg-Pro-Pro-Gly-Phe-Ser-Pro + Phe-Arg
show the reaction diagram
-
-
-
-
?
casein + H2O
?
show the reaction diagram
D-Val-Leu-Lys-4-nitroanilide + H2O
D-Val-Leu-Lys + 4-nitroaniline
show the reaction diagram
-
highest activity
-
-
?
extracellular matrix component type VII collagen
?
show the reaction diagram
-
-
-
-
-
factor Xa + H2O
?
show the reaction diagram
-
-
-
-
?
Fibrin + H2O
?
show the reaction diagram
Fibrin Aalpha-chain + H2O
?
show the reaction diagram
-
human, cleavage at Lys413-Lys414, the Bbeta-chain is cleaved more slowly and the gamma-chain is minimally affected
-
-
-
Fibrinogen + H2O
?
show the reaction diagram
fibrinogen + H2O
fibrin + ?
show the reaction diagram
fibrinogen + H2O
fibrin + propeptide
show the reaction diagram
Fibrinogen Aalpha-chain + H2O
?
show the reaction diagram
fibrinogen alpha chain + H2O
?
show the reaction diagram
-
-
-
-
?
Gelatin + H2O
?
show the reaction diagram
His-Thr-Glu-Ala-Leu-Val-Thr-Ser + H2O
?
show the reaction diagram
His-Thr-Glu-Arg-Leu-Val-Thr-Ser + H2O
?
show the reaction diagram
His-Thr-Glu-Asn-Leu-Val-Thr-Ser + H2O
?
show the reaction diagram
His-Thr-Glu-Glu-Leu-Val-Thr-Ser + H2O
?
show the reaction diagram
His-Thr-Glu-His-Leu-Val-Thr-Ser + H2O
?
show the reaction diagram
His-Thr-Glu-Leu-Leu-Val-Thr-Ser + H2O
?
show the reaction diagram
His-Thr-Glu-Lys-Ala-Val-Thr-Ser + H2O
?
show the reaction diagram
-
-
-
-
?
His-Thr-Glu-Lys-Arg-Val-Thr-Ser + H2O
?
show the reaction diagram
-
-
-
-
?
His-Thr-Glu-Lys-Asn-Val-Thr-Ser + H2O
?
show the reaction diagram
-
-
-
-
?
His-Thr-Glu-Lys-Glu-Val-Thr-Ser + H2O
?
show the reaction diagram
-
-
-
-
?
His-Thr-Glu-Lys-His-Val-Thr-Ser + H2O
?
show the reaction diagram
-
-
-
-
?
His-Thr-Glu-Lys-Leu-Val-Thr-Ser + H2O
?
show the reaction diagram
His-Thr-Glu-Lys-Phe-Val-Thr-Ser + H2O
?
show the reaction diagram
-
-
-
-
?
His-Thr-Glu-Lys-Pro-Val-Thr-Ser + H2O
?
show the reaction diagram
-
-
-
-
?
His-Thr-Glu-Lys-Ser-Val-Thr-Ser + H2O
?
show the reaction diagram
-
-
-
-
?
His-Thr-Glu-Phe-Leu-Val-Thr-Ser + H2O
?
show the reaction diagram
His-Thr-Glu-Pro-Leu-Val-Thr-Ser + H2O
?
show the reaction diagram
His-Thr-Glu-Ser-Leu-Val-Thr-Ser + H2O
?
show the reaction diagram
Insulin B-chain + H2O
?
show the reaction diagram
Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val + H2O
?
show the reaction diagram
low molecular weight kininogen + H2O
kallidin + ?
show the reaction diagram
low-molecular weight kininogen + H2O
kallidin + ?
show the reaction diagram
Nalpha-benzoyl-DL-arginine-4-nitroanilide + H2O
Nalpha-benzoyl-DL-arginine + 4-nitroaniline
show the reaction diagram
plasminogen + H2O
plasmin + ?
show the reaction diagram
pyroGlu-Gly-Arg-4-nitroanilide + H2O
pyroGlu-Gly-Arg + 4-nitroaniline
show the reaction diagram
-
-
-
-
?
S-2444 + H2O
?
show the reaction diagram
-
i.e. pyroGlu-Gly-Arg-4-nitroanilide
-
-
?
thrombin + H2O
?
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
bradykinin + H2O
Arg-Pro-Pro-Gly-Phe-Ser-Pro + Phe-Arg
show the reaction diagram
-
-
-
-
?
Fibrin + H2O
?
show the reaction diagram
Fibrinogen + H2O
?
show the reaction diagram
fibrinogen + H2O
fibrin + ?
show the reaction diagram
fibrinogen + H2O
fibrin + propeptide
show the reaction diagram
low molecular weight kininogen + H2O
kallidin + ?
show the reaction diagram
-
-
i.e. Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg
-
?
low-molecular weight kininogen + H2O
kallidin + ?
show the reaction diagram
-
initiates generation of bradykinin
-
-
?
plasminogen + H2O
plasmin + ?
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Calcium
-
potential binding site for calcium
Cu2+
-
34.2% increase of activity at 10 mM
Fe2+
-
76.7% increase of activity at 10 mM
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
4-amidinophenylmethylsulfonyl fluoride
-
5% residual activity at 2 mM
4-bromophenacyl bromide
-
78.5% residual activity at 2 mM
Al3+
-
29% residual activity at 5 mM
alpha2-Macroglobulin
-
Aprotinin
-
strong inhibition
Ca2+
-
strong inhibition
disodium-EDTA
-
21.1% residual activity at 4 mM
dithiothreitol
-
92.8% residual activity at 4 mM
Fe3+
-
36% residual activity at 5 mM
Hg2+
-
63% inhibition at 10 mM
iodoacetamide
-
49.9% residual activity at 4 mM
K+
-
30.9% inhibition at 10 mM
Mg2+
-
strong inhibition
Mn2+
-
strong inhibition
N-4-tosyl-phenylalanyl chloromethyl ketone
-
16.5% residual activity at 0.1 mM
-
Nalpha-tosyl-L-lysine chloromethylketone
-
51.5% residual activity at 0.1 mM
o-phenanthroline
-
-
Pb2+
-
85.5% inhibition at 10 mM
phenylmethylsulfonyl fluoride
SBTI
-
26% residual activity at 4 mM
-
SDS
-
29% residual activity at 0.01% (w/v)
tetraethylene pentamine
-
rapid and complete inhibition
Tetraethylenepentamine
Triton X-100
-
87% residual activity at 0.01% (v/v)
Tween 20
-
80% residual activity at 0.01% (v/v)
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0000654
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg
-
-
1.37
D-Val-Leu-Lys-4-nitroanilide
-
at pH 7.4 and 37°C
0.00000739
His-Thr-Glu-Ala-Leu-Val-Thr-Ser
-
-
0.00000338
His-Thr-Glu-Arg-Leu-Val-Thr-Ser
-
-
0.00000937
His-Thr-Glu-Asn-Leu-Val-Thr-Ser
-
-
0.00000164
His-Thr-Glu-Glu-Leu-Val-Thr-Ser
-
-
0.00000415
His-Thr-Glu-His-Leu-Val-Thr-Ser
-
-
0.00000528
His-Thr-Glu-Leu-Leu-Val-Thr-Ser
-
-
0.00000579 - 0.000579
His-Thr-Glu-Lys-Leu-Val-Thr-Ser
0.00000247
His-Thr-Glu-Lys-Phe-Val-Thr-Ser
-
-
0.00000113
His-Thr-Glu-Phe-Leu-Val-Thr-Ser
-
-
0.00000391
His-Thr-Glu-Pro-Leu-Val-Thr-Ser
-
-
0.00000832
His-Thr-Glu-Ser-Leu-Val-Thr-Ser
-
-
0.00000138
Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val
-
-
0.000000242
low molecular weight kininogen
-
-
-
additional information
additional information
-
analysis of kinetic and equilibrium binding parameters of streptokinase-plasmin catalytic complex formation and its regulation by effectors 6-AHA and benzamidine
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
6.03
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg
Agkistrodon contortrix
-
-
5
His-Thr-Glu-Ala-Leu-Val-Thr-Ser
Agkistrodon contortrix
-
-
1.97
His-Thr-Glu-Arg-Leu-Val-Thr-Ser
Agkistrodon contortrix
-
-
65.7
His-Thr-Glu-Asn-Leu-Val-Thr-Ser
Agkistrodon contortrix
-
-
0.46
His-Thr-Glu-Glu-Leu-Val-Thr-Ser
Agkistrodon contortrix
-
-
5.19
His-Thr-Glu-His-Leu-Val-Thr-Ser
Agkistrodon contortrix
-
-
0.16
His-Thr-Glu-Leu-Leu-Val-Thr-Ser
Agkistrodon contortrix
-
-
2.23
His-Thr-Glu-Lys-Leu-Val-Thr-Ser
Agkistrodon contortrix
-
-
0.2
His-Thr-Glu-Lys-Phe-Val-Thr-Ser
Agkistrodon contortrix
-
-
0.45
His-Thr-Glu-Phe-Leu-Val-Thr-Ser
Agkistrodon contortrix
-
-
1.59
His-Thr-Glu-Pro-Leu-Val-Thr-Ser
Agkistrodon contortrix
-
-
3.98
His-Thr-Glu-Ser-Leu-Val-Thr-Ser
Agkistrodon contortrix
-
-
0.09
Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val
Agkistrodon contortrix
-
-
0.0086
low molecular weight kininogen
Agkistrodon contortrix
-
-
-
additional information
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
additional information
-
native and PEG-bound enzyme, rapid interaction kinetics with inhibitor alpha2-macroglobulin
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
1200
-
purified PEG-bound enzyme, substrate azocasein
1260
-
purified native enzyme, substrate azocasein
10900
-
purified PEG-bound enzyme, substrate human fibrin
11300
-
purified native enzyme, substrate human fibrin
additional information
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 10
-
considerable activity is observed at pH 10.0 (84.4%). The enzyme exhibits poorer activity in the acidic pH 5.0-6.0 (53-64%)
6 - 10
-
more than 50% activity between pH 6.0 and 10.0
8.5 - 9.5
-
-
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40 - 60
-
considerable activity is observed at 60°C (88.6%) and at 40°C (68.6%)
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20800
-
1 * 20800, SDS-PAGE
22520
-
calculated molecular weight
22540
-
Agkistrodon contortrix, calculation from amino acid sequence
22750
-
Agkistrodon contortrix, isoform fib2, calculation from amino acid sequence
22880
-
Agkistrodon contortrix, isoform fib1, calculation from amino acid sequence
22890
-
calculation from amino acid sequence
23800
x * 23800, recombinant His-tagged alfimeprase, SDS-PAGE
25000
-
SDS-PAGE
42000
-
x * 42000, the enzyme exists in three isoforms of 42000, 48000 and 60000 Da, SDS-PAGE
43330
-
PEG-bound enzyme, gel filtration
55000
-
1 * 55000, SDS-PAGE
56043
-
1 * 56043, MALDI-TOF mass spectrometry
additional information
-
amino acid sequence
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
-
adduction of polyethylene glycol to reduce the rate of clearance from the circulation, and coupling to an Arg-Gly-Asp (RGD) like peptide imparting inhibitory activity on platelet aggregation and thrombus formation while maintaining full fibrinolytic activity
additional information
-
contains little or no carbohydrate
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 12
-
pre-incubation of fibrinolytic protease for 1 h at pH 4.0-12.0 causes substantial activity loss
733238
5.5
-
6 h, 30-35% loss of activity
31471
6.5 - 10
-
stable
31481
6.5
-
optimal pH for stability
31471
7 - 10
-
after 60 min incubation the enzyme possesses remarkable stability at pH 7.0-10.0 (98.5-100%). In the acidic pH (5.0-6.0), the enzyme retains 77 and 85% activity, respectively
733596
9
-
6 h, 45-50% loss of activity
31471
additional information
-
effect of pH on conformation
31481
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 80
-
fibrinolytic protease loses about 40-60% of its activity after 1 h incubation at 20-80°C
22
-
stable for 10 days
30 - 50
-
the enzyme is quite stable at 30-50°C for 60 min, but at 60°C and above activity decreases drastically
30 - 60
-
the enzyme remains stable between 30 and 60°C when incubated for 40 min
37
-
about 50% loss of activity after 2 days, complete loss of activity after 10 days
additional information
-
effect of temperature on conformation
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
Effect of zinc binding on the structure and stability of fibrolase
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, 20 mM Tris-HCl buffer, 3 months, 3% loss of enzyme activity
-
22°C, stable for 10 days
-
4°C or -20°C, stable for 6 months, 20-30% loss of activity after 1 year
-
4°C, 20 mM Tris-HCl buffer, 3 months, 5% loss of enzyme activity
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate precipitation and DEAE-Sephadex gel filtration
-
ammonium sulfate precipitation, DEAE-Sepharose column chromatography
-
ammonium sulfate precipitation, Hi Prep phenyl column chromatography, and Superdex 75 gel filtration
-
DEAE-Sepharose CL6B column chromatography, Sepharose CL4B column chromatography, and Mono Q HR column chromatography
-
high performance liquid chromatography, hydroxyapatite high performance liquid chromatography and cation exchange high performance liquid chromatography
-
HiTrap Q-Sepharose column chromatography, gel filtration
hydrophobic interaction high performance liquid chromatography, hydroxyapatite high performance liquid chromatography and cation exchange high performance liquid chromatography
-
hydrophobic interaction HPLC, hydroxyapatite HPLC and cation exchange HPLC
-
hydroxyapatite chromatography, cation exchange chromatography
-
immunoaffinity chromatography
-
native enzyme by hydrophobic interaction and hydroxyapatite chromatography
-
Q-Sepharose column chromatography, Sephacryl S-100 gel filtration, and Sephadex G-50 gel filtration
-
recombinant soluble His-tagged enzyme from Escherichia coli by nickel affinity and hydrophobic interaction chromatography to 95.6% purity
-
secreted recombinant His-tagged alfimeprase from Pichia pastoris strains of GS115 and KM71 cell culture medium by immunoaffinity chromatography to 96% purity
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
chimeric Arg-Gly-Asp-fibrolase is expressed in Escherichia coli BL21(DE3) cells
co-expression of the active signal-less His-tagged enzyme with chaperone DsbC in Escherichia coli cytoplasm, over 90% of the recombinant enzyme is soluble due to DsbC, while the chaperones FkpA and Skp do not have an effect on fibrolase solubility
-
expressed in Pichia pastoris
-
expression of the wild-type fibrolase and a truncated enzyme form, alfimeprase
-
gene alf, high level expression of His-tagged alfimeprase in Pichia pastoris strains of GS115 and KM71 by methanol induction, secretion of the recombinant enzyme to the cell culture medium
production of alfimeprase by recombinant DNA technology
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
cotton cake as nitrogen source enhances fibrinolytic protease production substantially (71%) as compared to control
soybean meal supports maximum protease production, followed by malt extract, cotton cake, gelatin and beef extract
the optimal conditions to obtain high protease production are 1.26% soybean flour and 1.23% glucose concentration
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
construction of a heterobifunctional chimeric derivative of fibrolase through linking covalently with a RGD-like peptide via a surface residue distant from the active site, structure, overview, the chimera shows fibrolase and platelet-binding ability, the chimeric enzyme exhibits an IC50 of 105 nM in inhibition of platelet aggregation, overview
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
using 50 mM Tris-HCl, 0.5 M L-arginine, 1 M NaCl, 0.5 mM ZnCl2, 1 mM oxidized glutathione, 2 mM reduced glutathione, and 2 M urea at pH 7.0. After the protein is added, the pH is adjusted to 7.5, and the protein is left to refold at 4°C for approximately 24 h
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
-
summary of results (including adverse events and pharmacodynamics) regarding drug development of alfimeprase as a potential drug against thrombosis and vascular disorders
medicine
pharmacology
-
alfimeprase is a potential therapeutic agents in thrombosis therapy, clinical indications and studies, overview