Information on EC 3.4.22.35 - Histolysain

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The expected taxonomic range for this enzyme is: Entamoeba

EC NUMBER
COMMENTARY hide
3.4.22.35
-
RECOMMENDED NAME
GeneOntology No.
Histolysain
-
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
hydrolysis of proteins, including basement membrane collagen and azocasein. Preferential cleavage: Arg-Arg-/- in small molecule substrates including Z-Arg-Arg-/-NHMec
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of peptide bond
CAS REGISTRY NUMBER
COMMENTARY hide
92228-52-9
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
gene edcp2 or acp2
-
-
Manually annotated by BRENDA team
strain HM 1:IMSS, catalytic classes of proteinases
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
Arg-Gly-Phe-Phe + H2O
Arg-Gly + Phe-Phe
show the reaction diagram
-
substrate of amoebapain
-
-
?
azocasein + H2O
?
show the reaction diagram
azocasein + H2O
fragments of azocasein
show the reaction diagram
Azocoll + H2O
?
show the reaction diagram
-
-
-
-
?
Azocoll + H2O
Hydrolyzed azocoll
show the reaction diagram
-
-
-
-
-
benzyloxycarbonyl-Ala-Arg-Arg-4-nitroanilide + H2O
benzyloxycarbonyl-Ala-Arg-Arg + 4-nitroaniline
show the reaction diagram
-
-
-
?
Benzyloxycarbonyl-Arg-Arg 4-methylcoumarin 7-amide + H2O
?
show the reaction diagram
-
-
-
-
-
benzyloxycarbonyl-Arg-Arg-4-nitroanilide + H2O
benzyloxycarbonyl-Arg-Arg + 4-nitroaniline
show the reaction diagram
benzyloxycarbonyl-Arg-Arg-7-amido-4-methylcoumarin + H2O
?
show the reaction diagram
-
-
-
?
benzyloxycarbonyl-L-arginyl-L-arginine 4-nitroanilide + H2O
benzyloxycarbonyl-L-arginyl-L-arginine + 4-nitroaniline
show the reaction diagram
-
-
?
Benzyloxycarbonyl-Phe-Arg 4-methylcoumarin 7-amide + H2O
?
show the reaction diagram
-
-
-
-
-
Benzyloxycarbonyl-Phe-L-citrullin 4-methylcoumarin 7-amide + H2O
?
show the reaction diagram
-
-
-
-
-
Bovine serum albumin + H2O
?
show the reaction diagram
-
-
-
?
C3 protein + H2O
?
show the reaction diagram
-
protein from human
-
-
?
Cartilage proteoglycan + H2O
?
show the reaction diagram
-
-
-
-
-
chemokine CCL13 + H2O
?
show the reaction diagram
chemokine CCL2 + H2O
?
show the reaction diagram
chemokine CXCL8 + H2O
?
show the reaction diagram
Collagen + H2O
?
show the reaction diagram
-
-
-
?
collagen type 1 + H2O
?
show the reaction diagram
-
-
-
-
?
Collagen type I + H2O
?
show the reaction diagram
-
digestion with an initial attack at the alpha2-chain
-
-
-
complement factor C3 + H2O
peptides
show the reaction diagram
-
product determination
?
complement factor C9 + H2O
peptides
show the reaction diagram
-
product determination
?
Fibronectin + H2O
?
show the reaction diagram
fibronectin + H2O
peptides
show the reaction diagram
-
product determination
?
Gelatin + H2O
?
show the reaction diagram
Hemoglobin + H2O
?
show the reaction diagram
-
-
-
?
hide powder azure + H2O
?
show the reaction diagram
-
-
-
-
-
immunoglobulin G + H2O
?
show the reaction diagram
immunoglobulin G + H2O
peptides derived from immunoglobulin G
show the reaction diagram
-
product determination
?
Kidney glomerular basement-membrane collagen + H2O
?
show the reaction diagram
-
-
-
-
-
Muc2 + H2O
?
show the reaction diagram
MUC2 mucin + H2O
?
show the reaction diagram
mucin + H2O
?
show the reaction diagram
mucin MUC2 polymer + H2O
?
show the reaction diagram
-
enzyme is involved, together with other Entamoeba histolytica cysteine proteinases, in the degradation of mucin in the mucous layer of the colon epithelium, altering the protective function to facilitate parasite attachment
-
?
N-benzyloxycarbonyl-L-arginyl-7-amido-4-methylcoumarin + H2O
N-benzyloxycarbonyl-L-arginine + 7-amino-4-methylcoumarin
show the reaction diagram
N-benzyloxycarbonyl-L-arginyl-L-arginyl-7-amido-4-methylcoumarin + H2O
N-benzyloxycarbonyl-L-arginyl-L-arginine + 7-amino-4-methylcoumarin
show the reaction diagram
N-benzyloxycarbonyl-L-phenylalanyl-L-arginyl-7-amido-4-methylcoumarin + H2O
N-benzyloxycarbonyl-L-phenylalanyl-L-arginine + 7-amino-4-methylcoumarin
show the reaction diagram
-
7% of the activity with N-benzyloxycarbonyl-L-arginyl-L-arginyl-7-amido-4-methylcoumarin
-
?
pro-interleukin-18 + H2O
?
show the reaction diagram
-
protein from human
-
-
?
protein + H2O
peptides
show the reaction diagram
Z-Ala-Arg-Arg-7-amido-4-methylcoumarin + H2O
Z-Ala-Arg-Arg + 7-amino-4-methylcoumarin
show the reaction diagram
Z-Arg-Arg-4-methoxy-2-nitroanilide + H2O
Z-Arg-Arg + 4-methoxy-2-nitroaniline
show the reaction diagram
-
-
-
-
?
Z-Arg-Arg-4-nitroanilide + H2O
Z-Arg-Arg + 4-nitroaniline
show the reaction diagram
-
-
-
-
?
Z-Arg-Arg-7-amido-4-methylcoumarin + H2O
Z-Arg-Arg + 7-amino-4-methylcoumarin
show the reaction diagram
Z-Arg-Arg-7-amido-4-trifluoromethylcoumarin + H2O
?
show the reaction diagram
-
-
-
-
?
Z-Arg-Arg-7-amido-4-trifluoromethylcoumarin + H2O
Z-Arg-Arg + 7-amino-4-trifluoromethylcoumarin
show the reaction diagram
-
-
-
?
Insulin B-chain + H2O
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
chemokine CCL13 + H2O
?
show the reaction diagram
-
Entamoeba histolytica is a human pathogen causing amoebic colitis, the enzyme is involved in the inflammation process modulating human host leucocyte migration by proteolytic cleavage of chemokines CCL2, CCL13, and CXCL8, a mechanism to circumvent host immune response
-
-
?
chemokine CCL2 + H2O
?
show the reaction diagram
-
Entamoeba histolytica is a human pathogen causing amoebic colitis, the enzyme is involved in the inflammation process modulating human host leucocyte migration by proteolytic cleavage of chemokines CCL2, CCL13, and CXCL8, a mechanism to circumvent host immune response
-
-
?
chemokine CXCL8 + H2O
?
show the reaction diagram
-
Entamoeba histolytica is a human pathogen causing amoebic colitis, the enzyme is involved in the inflammation process modulating human host leucocyte migration by proteolytic cleavage of chemokines CCL2, CCL13, and CXCL8, a mechanism to circumvent host immune response
-
-
?
collagen type 1 + H2O
?
show the reaction diagram
-
-
-
-
?
Fibronectin + H2O
?
show the reaction diagram
-
-
-
-
?
Muc2 + H2O
?
show the reaction diagram
MUC2 mucin + H2O
?
show the reaction diagram
-
disruption of the protective mucin layer of targeted host cells, entry mechanism of the pathogen, overview
-
-
?
mucin MUC2 polymer + H2O
?
show the reaction diagram
-
enzyme is involved, together with other Entamoeba histolytica cysteine proteinases, in the degradation of mucin in the mucous layer of the colon epithelium, altering the protective function to facilitate parasite attachment
-
?
protein + H2O
peptides
show the reaction diagram
additional information
?
-
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4-hydroxymercuribenzoate
-
E64 inhibits cell growth and is cytopathic/lethal for E64-sensitive and E64-resistant amoebae
4-[(2S,3S)-carboxyoxiran-2-ylcarbonyl-L-leucylamido]butylguanidine
-
i.e. trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane or E64
chicken cystatin
-
-
-
chymostatin
-
-
Cystatin
-
-
-
cysteine protease inhibitor 1
-
-
-
cysteine protease inhibitor 2
-
inhibits isoform CP5 approximately 20times more efficient than isoform CP2
-
EDTA
-
slight inhibition at 10 mM
EhICP1
Entamoeba histolytica inhibitor for cysteine protease type 1; Entamoeba histolytica inhibitor for cysteine protease type 1; Entamoeba histolytica inhibitor for cysteine protease type 1, inhibition also by regulation of CP5 processing
-
EhICP2
Entamoeba histolytica inhibitor for cysteine protease type 2; Entamoeba histolytica inhibitor for cysteine protease type 2; Entamoeba histolytica inhibitor for cysteine protease type 2, inhibition also by regulation of CP5 processing
-
iodoacetic acid
-
-
L-3-carboxy-2,3-trans-epoxypropionyl-leucyl-amido(4-guanidino)butane
-
i.e. E-64
L-trans-epoxysuccinyl-(ethoxy)-Leu-3-methylbutylamide ethyl ester
-
i.e. E64d, inhibits cell growth and is cytopathic/lethal for E64-sensitive and E64-resistant amoebae
L-trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane
-
-
L-trans-epoxysuccinyl-Leu-4-guanidinobutylamide
-
i.e. E64, inhibits cell growth in E64-resistant by about 80% and is cytopathic/lethal for E64-sensitive amoebae
N-ethylmaleimide
-
-
N-methylpiperazine-urea-phenylalanylhomoarginylvinylsulfone-benzene
-
WRR483, inhibitor addition to medium fully prevents invasion of parasite
N-methylpiperazine-urea-phenylalanylhomophenylalanylvinylsulfone-benzene
-
K11777, inhibitor addition to medium partially prevents invasion of parasite
Nalpha-4-tosyl-L-lysine chloromethylketone
-
-
Natural and synthetic inhibitors of cysteine proteinases
-
-
-
p-hydroxymercuribenzoate
-
Peptidyldiazomethanes
-
-
Phenanthroline
-
slight inhibition at 10 mM
tosylsulfonylleucylchloromethyl ketone
-
i.e. TLCK
tosylsulfonylphenylalanylchloromethyl ketone
-
i.e. TPCK
trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane
Z-Phe-Ala-diazomethylketone
-
E64 inhibits cell growth and is cytopathic/lethal for E64-sensitive and E64-resistant amoebae
Z-Phe-Phe-diazomethylketone
-
25% inhibition of E64-sensitive and E64-resistant amoba growth
Zn2+
-
77% inhibition of proteolytic activity at 2.5 mM, completely reversible by L-cystein, phenanthroline, and L-histidine, Zn2+ interferes with the fibronectin binding of the amoeba
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
-
stimulates
cysteine
-
5-10 mM activate refolded protein
dithiothreitol
Liposomes
association increases activity about 2fold
-
sulfhydryl compounds
-
activate
additional information
-
expression of ehpc1 and ehpc2 is increased in presence of E64 in E64-sensitive and E64-resistant amoebae, overview
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0015
benzyloxycarbonyl-Arg-Arg 4-methylcoumarin 7-amide
-
-
0.032
Benzyloxycarbonyl-Phe-Arg 4-methylcoumarin 7-amide
-
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
130
benzyloxycarbonyl-Arg-Arg 4-methylcoumarin 7-amide
Entamoeba histolytica
-
-
0.4
Benzyloxycarbonyl-Phe-Arg 4-methylcoumarin 7-amide
Entamoeba histolytica
-
-
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.132 - 0.809
cysteine protease inhibitor 1
0.05 - 0.95
cysteine protease inhibitor 2
0.00000013 - 0.00000081
EhICP1
0.00000005 - 0.00000095
EhICP2
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
170
-
purified native enzyme, substrate azocasein
180
-
purified recombinant enzyme, substrate azocasein
470
purified enzyme
890
purified enzyme associated to liposomes
1450
-
refolded, recombinant, activated enzyme
2800
-
purified enzyme
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.4
-
azocasein
5 - 6
-
dependent on the substrate
5.5 - 8
-
recombinant enzyme
5.5
-
azocasein
6
-
almost fully active between pH 5.5 and 7.5
6.7
-
hide powder azure
7
-
assay at, substrate azocasein
7.4
-
assay at
7.5
-
benzyloxycarbonyl-Phe-L-citrullin 4-methylcoumarin 7-amide, azocoll
9.5
-
benzyloxycarbonyl-Phe-Arg 4-methylcoumarin 7-amide, benzyloxycarbonyl-Arg-Arg 4-methylcoumarin 7-amide
additional information
-
neutral pH-optimum
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3.4 - 8.5
-
azocasein
4.5 - 9.5
-
recombinant enzyme
5 - 8
-
50% of activity at pH 5 and pH 8
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
at optimal pH, the turnover increases with increasing temperature up to 85C
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.7
-
isoelectric focusing
additional information
-
-
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
-
the enzyme is secreted and localized on the cell surface
Manually annotated by BRENDA team
additional information
-
bound to matrix-like structures both on the cell surface and within subcellular vesicles. The enzyme recycles between plasma membrane and pinocytic vesicles
-
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
29000
-
Entamoeba histolytica, gel filtration
66000
-
native PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
no glycoprotein
-
no N-glycosylation
proteolytic modification
-
the recombinant enzyme needs to be refolded and processed from the inactive precursor, ppEhCP112, to a 35.5 kDa mature and active enzyme, EhCP112. The thiol specific inhibitor E-64, but not serine or aspartic proteinase inhibitors arrests this activation process
additional information
-
processing of the inactive pro-enzyme to the mature active form by treatment with 10 mM DTT and 0.04% SDS at 37C and pH 8.8, SDS can be substituted by Triton X-100 and octylglucoside but these are 3fold less efficient
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 9.5
-
4C, stable during overnight incubation
30316
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4
-
pH 5.5-9.5, stable during overnight incubation
additional information
-
Hg2+, at low concentration causes a delay in heat denaturation
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
Hg2+, at low concentration causes a delay in heat denaturation
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
15fold, to homogeneity
-
Ni-Sepharose column chromatography
-
Ni2+-NTA resin column chromatography, and gel filtration
recombinant His-tagged enzyme from Escherichia coli
-
recombinant His-tagged full-length EhCP112 zymogen, containing the signal peptide and the pro-peptide, from Escherichia coli strain BL21 by nickel affinity chromatography
-
recombinant His-tagged inactive pro-enzyme to homogeneity
-
recombinant protein using His-tag under denaturating conditions
-
recombinant protein using His-tag under denaturating conditions; recombinant protein using His-tag under denaturating conditions; recombinant protein using His-tag under denaturating conditions
recombinant refolded His-tagged enzyme by nickel affinity chromatography
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DNA and amino acid sequence determination
expressed as His-tag and thioredoxin fusin protein in Escherichia coli
-
expressed as His-tag fusion protein in Escherichia coli BL21(DE3), formation of inclusion bodies; expressed as His-tag fusion protein in Escherichia coli BL21(DE3), formation of inclusion bodies; expressed as His-tag fusion protein in Escherichia coli BL21(DE3), formation of inclusion bodies
expressed in Escherichia coli BL21(DE3) cells
expressed in Escherichia coli C43 cells
-
expression of partial gene ehcp1 in Escherichia coli as GST-fusion protein, expression of functional EhCP2 in insect cells using the baculovirus transfection system
-
expression of the His-tagged inactive pro-enzyme in Escherichia coli BL21(DE3)
-
functional expression of EhCP5
-
gene ehcp112, functional expression of the His-tagged full-length EhCP112 zymogen, containing the signal peptide and the pro-peptide, in Escherichia coli strain BL21
-
gene Ehcp112, gene is encoded adjacent to Ehadh112, the latter encoding an adhesin that is covalently connected to the enzyme by electrostatic forces, DNA sequence determination and analysis, expression as His-tagged enzyme in Escherichia coli BL21(DE3)
-
genes ehcp1, ehcp2, and ehcp5, stable episomal transfection, overexpression of EhCP5 highly increases activity of EhCP1, EhCP2, and EhCP5 in recombinant amoeba and cytopathic activity, ehile overexpression of EhCP1 or 2 enhances only the single actvity and moderately enhances cytopathic activity, overview
-
recombinant expression of the His-tagged enzyme in Escherichia coli inclusion bodies
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
CP5 expression is significantly higher in the Entamoeba histolytica strain EGG isolated from liver than in the other strains maintained in culture (5.6fold higher than strain 32, 4.1fold higher than strain HM1 and 3.0fold higher than cultured strain EGG)
-
cysteine protease 6 is upregulated in response to heat shock and during pathogen invasion of the host tissue
cysteine protease 6 is upregulated in response to heat shock and during pathogen invasion of the host tissue; when Entamoeba histolytica is grown in the presence of E-64, PCP5 levels are significantly increased
-
-
when Entamoeba histolytica is grown in the presence of E-64, PCP5 levels are significantly increased
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
construction of EhCP5-deficient amoeba by antisense inhibition showing no activity on mucin from cells LS174T and HT-29F Cl.16E or against CHO cell layers, enzyme-deficient amoeba cannot overcome the mucus barrier to disrupt CHO cell monolayers, overview
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
2 mM SDS is the only surfactant that is effective at promoting the refolding and activation of the enzyme
-
dialysis against refolding buffer; dialysis against refolding buffer; dialysis against refolding buffer
recombinant His-tagged enzyme, solvation in 20 mM sodium phosphate, pH 7.4, 0.5 M NaCl, 10 mM imidazole, and 8 M urea, refolding and processing of the inactive precursor, ppEhCP112, to a 35.5 kDa mature and active enzyme, EhCP112
-
renaturation of purified inactive pro-enzyme by a 100fold dilution in a buffer containing reduced and oxidized thiols leading to a soluble inactive pro-enzyme
-
using 50 mM Tris pH 6.8,150 mM NaCl, 5% (v/v) glycerol, 0.5 mM oxidised glutathione, 3 mM reduced glutathione, at 4C