Information on EC 3.4.22.14 - actinidain

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The expected taxonomic range for this enzyme is: Actinidia

EC NUMBER
COMMENTARY hide
3.4.22.14
-
RECOMMENDED NAME
GeneOntology No.
actinidain
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Similar to that of papain
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of peptide bond
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY hide
39279-27-1
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
sarunashi or hardy kiwi
UniProt
Manually annotated by BRENDA team
Actinidia deliciosa cv. Hayward
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
-
active actinidin retains its primary structure and proteolytic activity after 2 h of simulated gastric digestion, followed by 2 h of intestinal digestion
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
2-(acetamido)ethylene 2'-pyridyl disulfide + H2O
?
show the reaction diagram
-
reactivity probe, analysis of temperature-dependence of reaction kinetics
-
-
?
2-(acetoxy)ethylene 2'-pyridyl disulfide + H2O
?
show the reaction diagram
-
reactivity probe, analysis of temperature-dependence of reaction kinetics
-
-
?
2-(N'-acetyl-D-phenylalanyl)hydroxyethylene 2'-pyridyl disulfide + H2O
?
show the reaction diagram
-
reactivity probe, analysis of temperature-dependence of reaction kinetics
-
-
?
2-(N'-acetyl-D-phenylalanylamino)ethylene 2'-pyridyl disulfide + H2O
?
show the reaction diagram
-
reactivity probe, analysis of temperature-dependence of reaction kinetics
-
-
?
2-(N'-acetyl-L-phenylalanyl)hydroxyethylene 2'-pyridyl disulfide + H2O
?
show the reaction diagram
-
reactivity probe, analysis of temperature-dependence of reaction kinetics
-
-
?
2-(N'-acetyl-L-phenylalanylamino)ethylene 2'-pyridyl disulfide + H2O
?
show the reaction diagram
-
reactivity probe, analysis of temperature-dependence of reaction kinetics
-
-
?
acetyl-Gly p-nitrophenyl ester + H2O
acetyl-Gly + 4-nitrophenol
show the reaction diagram
-
-
-
-
?
acetyl-Gly-Lys methyl ester + H2O
acetyl-Gly-Lys + methanol
show the reaction diagram
-
-
-
-
?
acetyl-Lys methyl ester + H2O
acetyl-Lys + methanol
show the reaction diagram
-
-
-
-
?
acid-soluble collagen + H2O
?
show the reaction diagram
-
-
-
-
?
benzoyl-Arg ethyl ester + H2O
benzoyl-Arg + ethanol
show the reaction diagram
-
-
-
-
?
benzoyl-Gly p-nitrophenyl ester + H2O
benzoyl-Gly + p-nitrophenol
show the reaction diagram
-
-
-
-
?
benzoyl-L-Arg ethyl ester + H2O
benzoyl-L-Arg ethanol
show the reaction diagram
-
-
-
-
?
benzyloxycarbonyl-Gly p-nitrophenyl ester + H2O
benzyloxycarbonyl-Gly + p-nitrophenol
show the reaction diagram
-
-
-
-
?
Benzyloxycarbonyl-Lys methyl ester + H2O
Benzyloxycarbonyl-Lys + methanol
show the reaction diagram
-
-
-
-
?
benzyloxycarbonyl-Lys p-nitrophenyl ester + H2O
benzyloxycarbonyl-Lys + 4-nitrophenol
show the reaction diagram
-
-
-
-
?
benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin + H2O
?
show the reaction diagram
butyloxycarbonyl-Phe-NH-CH2CN + H2O
?
show the reaction diagram
-
-
-
-
?
carboxybenzyl-L-Lys 4-nitrophenyl ester + H2O
carboxybenzyl-L-Lys + 4-nitrophenol
show the reaction diagram
-
-
-
-
?
casein + H2O
?
show the reaction diagram
Collagen + H2O
?
show the reaction diagram
Collagen type I + H2O
?
show the reaction diagram
collagen type II + H2O
?
show the reaction diagram
ethyl 2-pyridyl disulfide + H2O
?
show the reaction diagram
-
reactivity probe, analysis of temperature-dependence of reaction kinetics
-
-
?
Fibrinogen + H2O
?
show the reaction diagram
Gelatin + H2O
?
show the reaction diagram
Hemoglobin + H2O
?
show the reaction diagram
-
-
-
-
?
Insulin B-chain + H2O
?
show the reaction diagram
-
the enzyme cleaves most readily the carboxyl peptide bonds of those residues of the B-chain acylated by large hydrophobic residues - Val, Leu, or Phe, but not Tyr - and also the Arg22-Gly23 bond
-
-
-
kiwellin + H2O
KiTH + kissper
show the reaction diagram
-
in vitro treatment of purified kiwellin, an allergenic protein formerly isolated from green kiwi fruit, with the protease actinidin from green kiwi fruit originates KiTH, a 20 kDa protein with 100% identity with the C-terminal region of kiwellin, and kissper, a described pore-forming peptide
-
-
?
N-acetyl-L-Phe-Gly methyl ester + H2O
?
show the reaction diagram
-
stopped-flow spectral analysis of increasing content of co-solvent acetonitrile on kinetics
-
-
-
N-acetyl-Phe-Gly methyl thionoester + H2O
?
show the reaction diagram
-
-
-
-
?
N-alpha-benzyloxycarbonyl-L-lysine 4-nitrophenyl ester + H2O
N-alpha-benzyloxycarbonyl-L-lysine + 4-nitrophenol
show the reaction diagram
Nalpha-benzoyl-L-Arg p-nitroanilide + H2O
Nalpha-benzoyl-L-Arg + 4-nitroaniline
show the reaction diagram
-
-
-
-
?
Nalpha-benzyloxycarbonyl-L-Lys p-nitrophenyl ester + H2O
Nalpha-benzyloxycarbonyl-L-Lys + 4-nitrophenol
show the reaction diagram
-
-
-
-
?
occludin + H2O
?
show the reaction diagram
-
-
-
-
?
p-toluene-sulphonyl-L-glutamine p-nitrophenyl ester + H2O
p-toluene-sulphonyl-L-glutamine + 4-nitrophenol
show the reaction diagram
-
-
-
-
?
sheep IgG + H2O
?
show the reaction diagram
succinylcasein + H2O
?
show the reaction diagram
-
-
-
-
?
Tosyl-Arg methyl ester + H2O
Tosyl-Arg + methanol
show the reaction diagram
-
-
-
-
?
atelocollagen + 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
casein + H2O
?
show the reaction diagram
-
-
-
-
?
Gelatin + H2O
?
show the reaction diagram
-
-
-
-
?
kiwellin + H2O
KiTH + kissper
show the reaction diagram
-
in vitro treatment of purified kiwellin, an allergenic protein formerly isolated from green kiwi fruit, with the protease actinidin from green kiwi fruit originates KiTH, a 20 kDa protein with 100% identity with the C-terminal region of kiwellin, and kissper, a described pore-forming peptide
-
-
?
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1-(L-trans-epoxysuccinylleucylamino)-4-guanidinobutane
4,4'-dithiodipyridine
-
-
5,5'-dithiobis(2-nitrobenzoic acid)
-
-
Ba2+
-
addition leads to inhibition of free actinidin whereas immobilized actinidin shows a much weaker inhibition
Ca2+
-
addition leads to inhibition of free actinidin whereas immobilized actinidin shows a much weaker inhibition
Co2+
-
addition leads to inhibition of free actinidin whereas immobilized actinidin shows a much weaker inhibition
Cystatin
-
hybrids of chicken cystatin with human kininogen domain 2 sequences exhibit improved inhibition
-
cystatin C
-
recombinant inhibitor expressed in E. coli
-
Fe2+
-
addition leads to inhibition of free actinidin whereas immobilized actinidin shows a much weaker inhibition
human kininogen domain 2
-
hybrids of chicken cystatin with human kininogen domain 2 sequences exhibit improved inhibition
-
iodoacetate
-
-
KCl
-
minimal activity at 0.5-0.8 M, depending on substrate concentration. Minimal activity is 50-70% of the acitivity in absence of salt, with increase in KM-value and decrease in kcat-value. Slight reactivation above 0.8 M
L-kininogen
-
-
-
Leupeptin
-
-
LiCl
-
minimal activity at 1.0-1.5 M, depending on substrate concentration. Minimal activity is 50-70% of the acitivity in absence of salt, with increase in KM-value and decrease in kcat-value. Slight reactivation above 1.5 M
Mg2+
-
addition leads to inhibition of free actinidin whereas immobilized actinidin shows a much weaker inhibition
Mn2+
-
addition leads to inhibition of free actinidin whereas immobilized actinidin shows a much weaker inhibition
N-(trans-epoxysuccinyl)-L-leucine 4-guanidinobutylamide
N-acetyl-L-Arg
-
-
N-benzoyl-L-Arg
-
-
N-benzoyl-L-Arg ethyl ester
-
-
NaCl
-
minimal activity at 0.5-1.2 M, depending on substrate concentration. Minimal activity is 50-70% of the acitivity in absence of salt, with increase in KM-value and decrease in kcat-value. Slight reactivation above 1.2 M
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
dithiothreitol
-
activates
L-cysteine
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
78
acetyl-Lys methyl ester
-
-
61
benzoyl-Arg ethyl ester
-
-
0.31
benzoyl-Gly p-nitrophenyl ester
-
-
89
benzoyl-L-Arg ethyl ester
-
pH 5.6, 25C, in 0.3 M KCl
21
benzoyl-Lys methyl ester
-
-
0.12
benzyloxycarbonyl-Gly p-nitrophenyl ester
-
-
0.022
benzyloxycarbonyl-Lys p-nitrophenyl ester
-
-
0.044
butyloxycarbonyl-Phe-NH-CH2CN
-
-
-
0.0125 - 0.01592
casein
0.25 - 0.893
N-acetyl-L-Phe-Gly methyl ester
0.0582
N-alpha-benzyloxycarbonyl-L-lysine 4-nitrophenyl ester
-
pH 6.5, 27C, Vmax: 2.14 miroM/sec
-
1.8 - 11.2
Nalpha-benzoyl-L-Arg p-nitroanilide
0.0262
Nalpha-benzyloxycarbonyl-L-Lys p-nitrophenyl ester
-
-
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.7
acetyl-Lys methyl ester
Actinidia chinensis
-
-
4.5
benzoyl-Arg ethyl ester
Actinidia chinensis
-
-
0.029
benzoyl-Arg p-nitroanilide
Actinidia chinensis
-
-
5.8
benzoyl-Gly p-nitrophenyl ester
Actinidia chinensis
-
-
2.6
benzoyl-L-Arg ethyl ester
Actinidia chinensis
-
pH 5.6, 25C, in 0.3 M KCl
3.4
benzyloxycarbonyl p-nitrophenyl ester
Actinidia chinensis
-
-
6
Benzyloxycarbonyl-Lys methyl ester
Actinidia chinensis
-
-
29
benzyloxycarbonyl-Lys p-nitrophenyl ester
Actinidia chinensis
-
-
20.74 - 29.2
casein
42.8
N-alpha-benzyloxycarbonyl-L-lysine 4-nitrophenyl ester
Actinidia deliciosa
-
pH 6.5, 27C
-
33
Nalpha-benzyloxycarbonyl-L-Lys p-nitrophenyl ester
Actinidia chinensis
-
-
additional information
additional information
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1300 - 2330
casein
730
N-alpha-benzyloxycarbonyl-L-lysine 4-nitrophenyl ester
Actinidia deliciosa
-
pH 6.5, 27C
206877
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.35
KCl
-
25C, pH 6.5
0.13
LiCl
-
25C, pH 6.5
0.43
NaCl
-
25C, pH 6.5
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.033
-
isoenzyme KP2
0.034
-
isoenzyme KP3
0.04
-
isoenzyme KP1
0.051
-
isoenzyme KP5
0.177
-
isoenzyme KP6
0.197
-
isoenzyme KP4
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 7
-
benzoyl-L-Arg ethyl ester
5.5
-
efficient proteolytic activity towards casein
6.5
-
assay at
7
-
optimal temperature, free actinidin
7.2
-
assay at
8.5
-
assay at; optimal temperature, immobilized actinidin
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3.5 - 8.8
-
about 45% of maximal activity at pH 3.5 and at pH 8.8
7 - 8.5
efficient digestion of type I and type II collagen
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20
-
assay at
27
-
assay at
40
-
optimal temperature, free actinidin
55
-
assay at
60
-
optimal temperature, immobilized actinidin
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
actinidin is present in small cells, but not large cells in the outer pericarp of mature Actinidia deliciosa fruit at harvest. Within the small cells, actinidin is localised diffusely in the vacuole, associated with the plasma membrane, and in a layer in the plastids near starch granules
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
-
actinidin is present in small cells, but not large cells in the outer pericarp of mature Actinidia deliciosa fruit at harvest. Within the small cells, actinidin is localised diffusely in the vacuole, associated with the plasma membrane, and in a layer in the plastids near starch granules
Manually annotated by BRENDA team
-
actinidin is present in small cells, but not large cells in the outer pericarp of mature Actinidia deliciosa fruit at harvest. Within the small cells, actinidin is localised diffusely in the vacuole, associated with the plasma membrane, and in a layer in the plastids near starch granules
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
12800
-
gel filtration
23000
-
SDS-PAGE
23880
-
MALDI-TOF
26000
-
high speed equilibrium method
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging drop vapor diffusion method, using 30 mM CdCl2 and 30% (w/v) PEG 400 in 0.1 M sodium acetate pH 4.6, at 15C
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2.1
-
1-2 s, 40% of the catalytic sites survive
81475
2.4
-
1-2 s, 75% of the catalytic sites survive
81475
2.5
-
above, 1-2 s, stable
81475
3 - 12
-
immobilized actinidin is more stable than free form at pH 3 and pH 12 after 60 min incubation leading to 30% residual activity of immobilized form and complete inactivation of the free form
733988
additional information
-
thermal stability of Act d 1 and Act d 2 is strongly pH dependent. Act d 1 is irreversibly destabilized in acidic solutions (pH 3.5)
700290
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
70
-
half life: 12 min free actinidin
80
-
half life: 4 min free actinidin, 32 min immobilized actinidin
95
-
the enzyme is thermally inactivated after 5 min at 95C
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
active actinidin retains its primary structure and proteolytic activity after 2 h of simulated gastric digestion, followed by 2 h of intestinal digestion
-
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
acetonitrile
-
stopped-flow spectral analysis of increasing content of co-solvent acetonitrile on kinetics. With low acetonitrile content, plots of reaction are linear, increasing the acetonitrile content results in marked deviations of the plots from linearity with a rate minimum around [S]0 of 0.15 mM
dimethyl sulfoxide
-
comparison of effect on reaction kinetics with acetonitrile
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
inactivated by methylene blue-catalyzed photooxidation, at pH 7.9 and 20C. The rate of inactivation is pH-dependent and becomes slower at lower pH values
-
81476
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
20C, 30 days, free actinidin shows no activity while immobilized actinidin shows 90% activity
-
4C, 60 days, free actinidin shows no activity while immobilized actinidin shows 80% activity
-
4C, crystalline enzyme, resuspended in dialysis buffer at pH 4, stable for several months
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; the enzyme fraction is precipitated from kiwifruit extract by 60% saturation of ammonium sulfate. The precipitate is redissolved in 50 mM citrate buffer (pH 5.5) and dialyzed overnight against this buffer. The dialyzate is loaded into a DEAE-Sepharose Fast Flow column, which pre-equilibrated with the same buffer. The adsorbed fractions are eluted with 0.0-1 M linear gradient of sodium chloride in the buffer
Act d 1 is purified from kiwifruit extracts by covalent chromatography using activated thiopropyl sepharose 6B
-
actinidin can be separated into six proteases, named KP1, KP2, KP3, KP4, KP5 and KP6
-
Actinidin is purified from the soluble fraction of kiwi fruit by ion exchange chromatography on DE-52 and Mono-Q columns
-
ammonium sulfate precipitation and DEAE-Sephadex gel filtration
-
DEAE-Sephadex A-50 gel filtration and SP-Sephadex C-50 gel filtration
-
multiple forms
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Nicotiana benthamiana T1, T2, T3, and T4 transgenic lines
expression in Escherichia coli as inclusion bodies
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
P75Q
-
the mutant shows reduced activity compared tot he wild type enzyme
proteolytic modification
-
the enzyme is secreted in an inactive form as zymogen, named actininidin, which has a MW of 39000 Da
S68G
-
inactive
S68G/P75Q
-
the mutant shows reduced activity compared tot he wild type enzyme
P75Q
-
the mutant shows reduced activity compared tot he wild type enzyme
S68G
-
inactive
S68G/P75Q
-
the mutant shows reduced activity compared tot he wild type enzyme
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
-
actinidin might be utilized to eliminate the milk fat globule membranes (MFGM) protein residues from cream and its derivatives
food industry
-
actinidin is used as a beef tenderizer, use of actinidin-tenderized beef significantly improves emulsion stability, texture, and organoleptic properties of the sausage product
medicine
molecular biology
actinidin compared with type II or IV collagenase isolates intact human umbilical vein endothelial cells, hepatocytes, and thymic epithelial cells with viability more than 90%
nutrition
-
actinidin induces protease-dependent morphology changes of T84 human colorectal adenocarcinoma cells leading to cell rounding and desquamation of the epithelial monolayer, without affecting cell viability
synthesis
the enzyme can be used efficiently for hydrolysis of collagen and isolation of different cell populations from various solid tissues