Information on EC 3.4.22.30 - Caricain

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The expected taxonomic range for this enzyme is: Carica papaya

EC NUMBER
COMMENTARY
3.4.22.30
-
RECOMMENDED NAME
GeneOntology No.
Caricain
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
hydrolysis of proteins with broad specificity for peptide bonds, similar to those of papain and chymopapain
show the reaction diagram
-
-
-
-
hydrolysis of proteins with broad specificity for peptide bonds, similar to those of papain and chymopapain
show the reaction diagram
mechanism, Asp158 is involved in catalysis
-
hydrolysis of proteins with broad specificity for peptide bonds, similar to those of papain and chymopapain
show the reaction diagram
Asp158 is not involved in ionization and catalytic competence
-
hydrolysis of proteins with broad specificity for peptide bonds, similar to those of papain and chymopapain
show the reaction diagram
residue E50 is part of the electrostatic switch required for catalytic activity, mechanism, possible role of D158
-
hydrolysis of proteins with broad specificity for peptide bonds, similar to those of papain and chymopapain
show the reaction diagram
mechanism
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
hydrolysis
-
-
hydrolysis of peptide bond
-
-
-
-
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
caricain
P10056
-
Papaya peptidase A
-
-
-
-
Papaya peptidase II
-
-
-
-
Papaya proteinase 3
-
-
-
-
Papaya proteinase A
-
-
-
-
Papaya proteinase III
-
-
-
-
Papaya proteinase OMEGA
-
-
-
-
Papaya proteinase OMEGA
-
-
Papaya proteinase OMEGA
P10056
-
PPIII
-
-
-
-
PpOmega
-
-
-
-
procaricain
-
-
Proteinase omega
-
-
-
-
Proteinase, papaya A
-
-
-
-
Proteinase, papaya, III
-
-
-
-
Chymopapain S
-
-
-
-
additional information
-
names and numbers of papaya proteinases
additional information
-
problems of classification of papaya latex proteinases
CAS REGISTRY NUMBER
COMMENTARY
39307-22-7
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
commercial chymopapain
-
-
Manually annotated by BRENDA team
isoform caricain II
-
-
Manually annotated by BRENDA team
purified enzyme
-
-
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
Albumin + H2O
?
show the reaction diagram
-
low activity
-
-
-
alpha-N-benzoyl-L-arginine 4-nitroanilide + H2O
alpha-N-benzoyl-L-arginine + 4-nitroaniline
show the reaction diagram
-
-
-
?
azocasein + H2O
?
show the reaction diagram
-
-
-
-
-
Benzyloxycarbonyl-Gly 4-nitrophenyl ester + H2O
?
show the reaction diagram
-
-
-
-
-
Benzyloxycarbonyl-Lys nitrophenyl ester + H2O
?
show the reaction diagram
-
-
-
-
-
Benzyloxycarbonyl-Phe-Arg 4-methylcoumarin 7-amide + H2O
?
show the reaction diagram
-
-
-
-
-
Bovine nasal cartilage + H2O
?
show the reaction diagram
-
-
-
-
-
casein + H2O
?
show the reaction diagram
-
beta-casein, less active than papain
-
-
-
casein + H2O
?
show the reaction diagram
-
less active than papain
-
-
-
casein + H2O
?
show the reaction diagram
-
isoform caricain II, twofold activity on casein compared to caricain
-
-
-
Gliadin + H2O
?
show the reaction diagram
-
a rat liver lysosome assay is used to monitor the extent of detoxification of a gliadin digest by caricain. Pre-incubating the gliadin digest for different durations with caricain allows the kinetics of the detoxification process to be studied. A significant degree of protection (80%) of the lysosomes is achieved with 1.7% w/w of caricain on substrate after incubation for 2 h at 37 °C. The detoxification follows first-order kinetics with a rate constant of 0.00017/sec
-
-
?
gliadin + H2O
fragments of gliadin
show the reaction diagram
-
-
-
-
?
hide powder azure + H2O
?
show the reaction diagram
-
-
-
-
-
Hippuric acid 4-nitrophenyl ester + H2O
?
show the reaction diagram
-
-
-
-
-
N-acetyl-Phe-Gly methylthionoester + H2O
?
show the reaction diagram
-
synthetic chromogenic substrate, reaction mechanism, computer modeling of intermediate formation
-
?
N-Benzoyl-Arg 4-nitroanilide + H2O
?
show the reaction diagram
-
-
-
-
-
N-Benzoyl-Arg 4-nitroanilide + H2O
?
show the reaction diagram
-
less active than papain
-
-
-
N-Benzoyl-Arg amide + H2O
?
show the reaction diagram
-
less active than papain
-
-
-
N-Benzoyl-L-Arg ethyl ester + H2O
?
show the reaction diagram
-
-
-
-
-
N-Benzoyl-L-Arg ethyl ester + H2O
?
show the reaction diagram
-
less active than papain
-
-
-
N-benzoyl-L-arginine ethyl ester + H2O
N-benzoyl-L-arginine + ethanol
show the reaction diagram
-
isoform caricain II, low activity
-
-
?
N-tosyl-L-Arg methyl ester + H2O
N-tosyl-L-Arg + methanol
show the reaction diagram
-
less active than papain
-
-
-
protein + H2O
peptides
show the reaction diagram
-
-
-
?
protein + H2O
peptides
show the reaction diagram
-
-
-
?
protein + H2O
peptides
show the reaction diagram
-
-
-
?
protein + H2O
peptides
show the reaction diagram
-
-
-
?
protein + H2O
peptides
show the reaction diagram
-
-
-
?
protein + H2O
peptides
show the reaction diagram
-
-
-
?
protein + H2O
peptides
show the reaction diagram
-
-
-
?
protein + H2O
peptides
show the reaction diagram
-, P10056
-
-
?
pyr-Glu-Phe-Leu-4-nitroanilide + H2O
pyr-Glu-Phe-Leu + 4-nitroaniline
show the reaction diagram
-, P10056
-
-
?
Succinyl-albumin + H2O
?
show the reaction diagram
-
-
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
gliadin + H2O
fragments of gliadin
show the reaction diagram
-
-
-
-
?
protein + H2O
peptides
show the reaction diagram
-
-
-
?
protein + H2O
peptides
show the reaction diagram
-
-
-
?
protein + H2O
peptides
show the reaction diagram
-
-
-
?
protein + H2O
peptides
show the reaction diagram
-
-
-
?
protein + H2O
peptides
show the reaction diagram
-
-
-
?
protein + H2O
peptides
show the reaction diagram
-
-
-
?
protein + H2O
peptides
show the reaction diagram
-
-
-
?
protein + H2O
peptides
show the reaction diagram
-, P10056
-
-
?
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
Benzyloxycarbonyl-Phe-Arg 4-methylcoumarin 7-amide
-
competitive to succinyl-albumin
Gly-Gly(O-benzyl)Tyr-Arg
-
competitive, affinity is dependent on ionic strength and always lower than that of papain
imidazole
-
-
iodoacetamide
-
-
phenylmethylsulfonyl fluoride
-
weak inhibition
Succinyl-albumin
-
competitive to benzyloxycarbonyl-Phe-Arg 4-methylcoumarin 7-amide
-
trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane
-
i.e. E-64, irreversible, active site-directed
ACTIVATING COMPOUND
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
thiol group
-
enzyme contains 1 thiol group per molecule, this thiol group is essential for catalytic activity
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
16
-
benzoyl-DL-Arg 4-nitroanilide
-
pH 9.2
18
-
benzoyl-DL-Arg 4-nitroanilide
-
pH 4.0
0.028
-
benzyloxycarbonyl-Lys nitrophenyl ester
-
pH 5.9
0.035
-
benzyloxycarbonyl-Lys nitrophenyl ester
-
-
0.009
-
N-Benzyloxycarbonyl-Gly 4-nitrophenyl ester
-
-
0.000096
-
Succinyl-albumin
-
-
-
0.0067
-
Benzyloxycarbonyl-Phe-Arg 4-methylcoumarin 7-amide
-
-
additional information
-
additional information
-
kcat/Km ratio (pH-dependence of)
-
additional information
-
additional information
-
kcat/Km ratio; kinetic analysis
-
additional information
-
additional information
-
kinetic analysis
-
additional information
-
additional information
-
kinetics
-
additional information
-
additional information
-
kinetics of the reaction with substrate N-acetyl-Phe-Gly methylthionoester
-
additional information
-
additional information
-
kinetics, pH-dependence
-
additional information
-
additional information
-
kinetics
-
additional information
-
additional information
-
kinetics of wild-type and D158N mutant
-
additional information
-
additional information
-
kinetics
-
additional information
-
additional information
-
-
-
TURNOVER NUMBER [1/s]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.14
-
benzoyl-DL-Arg 4-nitroanilide
-
pH 4.0
0.15
-
benzoyl-DL-Arg 4-nitroanilide
-
pH 9.2
49
-
benzyloxycarbonyl-Lys nitrophenyl ester
-
pH 5.3 and 5.9
16.7
-
N-Benzyloxycarbonyl-Gly 4-nitrophenyl ester
-
-
2.6
-
Succinyl-albumin
-
-
-
7.1
-
Benzyloxycarbonyl-Phe-Arg 4-methylcoumarin 7-amide
-
-
additional information
-
additional information
-
pH-dependence of kcat/Km ratio
-
additional information
-
additional information
-
-
-
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
6.8
-
-
-
6.8
-
-
enzyme is dissolved in phosphate buffer and used with a suspension of lysosomes in 0.154 mol/l sodium chloride
additional information
-
-
pI: 11.0
additional information
-
-
papaya peptidase A is an enzyme of extreme basicity and has a higher isoelectric point than any of the other enzymes in papaya latex
additional information
-
-
pH profile
additional information
-
-
pH profiles of wild-type and mutant enzymes
pH RANGE
pH RANGE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
3
9.5
-
-
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
25
-
-
assay at
37
-
-
assay at
additional information
-
-
determination of minimum energy conformation, interaction of ionized and un-ionized side chains of Asp158 and their microenvironment
additional information
-
-
thermal transition state determination
SOURCE TISSUE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
SOURCE
-
non-fruit
Manually annotated by BRENDA team
-
latex from ripe and unripe fruit. Repeated wounding results in either accumulation or activation of enzyme as well as papain, chymopapain and more cysteine proteases
Manually annotated by BRENDA team
-
; estimated as a fraction of papaya latex
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
23500
-
-
-
24000
-
-
Carica papaya, sedimentation equilibrium centrifugation
40000
-
-
procaricain, fractions are estimated from plots of Kav against molecular weight using dextran blue, human gamma-globulin, bovine serum albumin and trypsin inhibitor as standards, mass spectrometric analysis of trypsin-digested fractions from chromatography are carried out by liquid chromatography/electrospray quadrupole time-of-flight mass spectrometry in positive mode
additional information
-
-
primary structure
additional information
-
-
three-dimensional structure deduced by knowledge-based modelling and active-centre characteristics determined by two-hydronic-state reactivity probe kinetics and kinetics of catalysis
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
?
-
x * 18800, purified from enzyme solution. Protein lacks the N-terminal 41 residues of enzyme, disulfide bond C22-C63 is opened
monomer
-
1 * 24000, Carica papaya, SDS-PAGE
additional information
-
transition state structure
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
additional information
-
enzyme is synthesized as inactive proenzyme, and rapidly converted to the active from within 2 min after wounding of the plant
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
pH STABILITY
pH STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
2
3
-
enzyme undergoes conformational transition that instantaneously converts the native form into a molten globule state and is completely irreversible
TEMPERATURE STABILITY
TEMPERATURE STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
85
-
-
heat stability of caricain up to 85°C
additional information
-
-
denaturation occurs as a non two-state process, via an intermediate structure, thermodynamic characteristics
additional information
-
-
thermal denaturing kinetics, autolysis during denaturation is prevented by irreversible blockage of the active-site thiol with idoacetamide
STORAGE STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
4°C, preparation of the mercury derivative of fully active papaya peptidase A, 50fold molar excess of DTT, 30 days stable
-
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
enzyme is semi-purified with from latex crude extracts by ion exchange chromatography on a CM Sephadex column C-50 and gel filtration
-
recombinant wild-type and mutant enzymes from Escherichia coli, to homogeneity
-
to homogeneity
-
to homogeneity, several ligands possible for affinity chromatography are presented, fractionation on hydrophobic and cation-exchange supports, overview
-
with monoclonal antibodies
-
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
overexpression of wild-type and mutant enzymes in Escherichia coli BL21(DE3)
-
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
D158N
-
altered ionization kinetics required for catalysis
D158N
-
site-directed mutagenesis, reduced activity at pH 6.8
E50A
-
site-directed mutagenesis, reduced activity at pH 6.8
E50A/D158N
-
site-directed mutagenesis, reduced activity at pH 6.8
Renatured/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
recombinant wild-type and mutant enzymes from inclusion bodies after overexpression in scherichia coli
-
APPLICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
drug development
-
caricain would be suitable for enzyme therapy in gluten intolerance and appears to have synergistic action with porcine intestinal extracts, potential for enzyme therapy in coeliac disease
nutrition
-
at low pH, enzyme undergoes conformational transition leading to instability and rapid degradation by pepsin. To be effective in gut after oral administration, enzyme needs to be protected against acid denaturation and degradation