Information on EC 3.4.23.12 - nepenthesin

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The expected taxonomic range for this enzyme is: Eukaryota

EC NUMBER
COMMENTARY
3.4.23.12
-
RECOMMENDED NAME
GeneOntology No.
nepenthesin
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Similar to pepsin, but also cleaves on either side of Asp and at Lys-/-Arg
show the reaction diagram
splits peptide bonds at carboxyl and amino sides of aspartic acid residue, at carboxyl side of alanine and probably carboxyl side of lysine
-
Similar to pepsin, but also cleaves on either side of Asp and at Lys-/-Arg
show the reaction diagram
predominantly specific for aspartic acid residues at its carboxyl side or at its amino side, also to tyrosine and alanine residues at carboxyl sides
Nepenthes sp.
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
hydrolysis of peptide bond
-
-
endopeptidase
-
SYNONYMS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
nepenthacin
-
-
-
-
nepenthasin;aspartyl endopeptidase
-
-
-
-
nepenthes aspartic protease
-
-
-
-
Nepenthesin
-
-
-
-
nepenthesin I
-
isoform
nepenthesin I
-
-
nepenthesin I
-
isoform
nepenthesin II
-
-
nepenthesin IIa
-
isoform
nepenthesin IIb
-
isoform
proteinase, nepenthes acid
-
-
-
-
EC 3.4.99.4
-
-
formerly
-
additional information
-
the enzyme belongs to the A1 peptidase family
CAS REGISTRY NUMBER
COMMENTARY
9073-80-7
-
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
subsp. flavescens
-
-
Manually annotated by BRENDA team
isozymes nepenthesin I and II
-
-
Manually annotated by BRENDA team
Nepenthes dormaniana
-
-
-
Manually annotated by BRENDA team
isozymes nepenthesin I and II
-
-
Manually annotated by BRENDA team
Nepenthes mixta
-
-
-
Manually annotated by BRENDA team
Nepenthes neufvilleana
-
-
-
Manually annotated by BRENDA team
Nepenthes sp.
-
-
-
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
acid-denatured haemoglobin + H2O
?
show the reaction diagram
-
-
-
?
Bovine hemoglobin + H2O
?
show the reaction diagram
-
-
-
-
?
casein + H2O
?
show the reaction diagram
Nepenthes sp.
-
-
-
-
?
casein + H2O
?
show the reaction diagram
-
-
-
-
?
Egg albumin + H2O
?
show the reaction diagram
-
-
-
-
?
EQAGGDATEDFEDVGHSTDAR + H2O
EQAGGDATED + FEDVGHST + DAR
show the reaction diagram
Nepenthes sp.
-
-
-
?
Fibrin + H2O
?
show the reaction diagram
-
-
-
-
?
FVNQHLCGSHLVEALYLVCGERGFFYTPKA + H2O
FVNQHL + CGSHLVE + Ala-Leu + Tyr + LVCGERGF + FYTPKA
show the reaction diagram
-
substrate is the insulin B chain
-
-
?
FVNQHLCGSHLVEALYLVCGERGFFYTPKA + H2O
FVNQHL + CGSHLVE + Ala-Leu + Tyr + LVCGERGF + FYTPKA
show the reaction diagram
-
substrate is the oxidized insulin B chain, cleavage by nepenthesin I site specificity, overview
-
-
?
Hemoglobin + H2O
?
show the reaction diagram
-
-
-
-
?
Hemoglobin + H2O
?
show the reaction diagram
-
acid-denatured protein substrate
-
-
?
KDEAASDVK + H2O
KDEAAS + DVK
show the reaction diagram
Nepenthes sp.
-
-
-
?
KIYKDTEGY + H2O
KIY + KD + TEGY
show the reaction diagram
Nepenthes sp.
-
-
-
?
LFNQDVDAAVR + H2O
LFNQDVD + AAVR
show the reaction diagram
Nepenthes sp.
-
-
-
?
LFNQDVDAAVR + H2O
LFNQDVD + AAVR
show the reaction diagram
Nepenthes sp.
-
-
-
-
?
LKPVYDSLDAVRR + H2O
LKPVY + DSL + DAVRR
show the reaction diagram
Nepenthes sp.
-
-
-
?
NQDVDAAVRGIK + H2O
NQDV + L-Asp + AAVRGIK
show the reaction diagram
Nepenthes sp.
-
-
-
?
oxidized insulin B chain + H2O
?
show the reaction diagram
-
nepenthesin I: 80% cleavage of Phe24-Phe25, 67% cleavage of Glu13-Ala14, 50% cleavage of Leu6-cysteic-acid7, 38% cleavage of Leu15-Tyr16 and 33% cleavage of Tyr16-Leu17
-
?
Peptides + H2O
?
show the reaction diagram
-
preferential cleavage: -Asp, Asp-, Lys-, Ala-
-
-
-
Peptides + H2O
?
show the reaction diagram
-
preferential cleavage: -Asp, Asp-, Lys-, Ala-
-
-
-
Peptides + H2O
?
show the reaction diagram
-
preferential cleavage: -Asp, Asp-, Lys-, Ala-
-
-
-
Peptides + H2O
?
show the reaction diagram
Nepenthes sp.
-
preferential cleavage: -Asp, Asp-, Lys-, Ala-
-
-
-
Peptides + H2O
?
show the reaction diagram
-
preferential cleavage: -Asp, Asp-, Lys-, Ala-
-
-
-
Peptides + H2O
?
show the reaction diagram
-
preferential cleavage: -Asp, Asp-, Lys-, Ala-
-
-
-
Peptides + H2O
?
show the reaction diagram
Nepenthes dormaniana, Nepenthes neufvilleana
-
preferential cleavage: -Asp, Asp-, Lys-, Ala-
-
-
-
Peptides + H2O
?
show the reaction diagram
-
preferential cleavage: -Asp, Asp-, Lys-, Ala-
-
-
-
Peptides + H2O
?
show the reaction diagram
Nepenthes mixta
-
preferential cleavage: -Asp, Asp-, Lys-, Ala-
-
-
-
Ribonuclease + H2O
?
show the reaction diagram
-
-
-
-
?
TGTWDAYK + H2O
TGTWDA + YK
show the reaction diagram
Nepenthes sp.
-
-
-
?
TGTYDATK + H2O
TGTYDA + T-K
show the reaction diagram
Nepenthes sp.
-
-
-
?
Thr-Ile-Asp-Glu + H2O
Thr-Ile-Asp + Glu
show the reaction diagram
Nepenthes sp.
-
-
-
?
WDEAVNLAK + H2O
WDEA + VNLAK
show the reaction diagram
Nepenthes sp.
-
-
-
?
LQQKRWDEAA + H2O
LQQK + RW + DEAA
show the reaction diagram
Nepenthes sp.
-
-
-
?
additional information
?
-
-
the enzyme is involved in digestion of prey, mainly insects, for nitrogen uptake of the carnivorous plant, and is therefore secreted into the pitcher fluid
-
-
-
additional information
?
-
-
cleavage site specificity of nepenthesin I and II, overview
-
-
-
additional information
?
-
-
cleavage site specificity with preference for the carboxy and amino sides of Asp, cleavage of the carboxy side of Ala, Leu, Ser, Thr, and Tyr also occurs, and cleavage on the amino side of Ala, Phe, Thr, Tyr, and Lys
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
Peptides + H2O
?
show the reaction diagram
-
preferential cleavage: -Asp, Asp-, Lys-, Ala-
-
-
-
Peptides + H2O
?
show the reaction diagram
-
preferential cleavage: -Asp, Asp-, Lys-, Ala-
-
-
-
Peptides + H2O
?
show the reaction diagram
-
preferential cleavage: -Asp, Asp-, Lys-, Ala-
-
-
-
Peptides + H2O
?
show the reaction diagram
Nepenthes sp.
-
preferential cleavage: -Asp, Asp-, Lys-, Ala-
-
-
-
Peptides + H2O
?
show the reaction diagram
-
preferential cleavage: -Asp, Asp-, Lys-, Ala-
-
-
-
Peptides + H2O
?
show the reaction diagram
-
preferential cleavage: -Asp, Asp-, Lys-, Ala-
-
-
-
Peptides + H2O
?
show the reaction diagram
Nepenthes dormaniana, Nepenthes neufvilleana
-
preferential cleavage: -Asp, Asp-, Lys-, Ala-
-
-
-
Peptides + H2O
?
show the reaction diagram
-
preferential cleavage: -Asp, Asp-, Lys-, Ala-
-
-
-
Peptides + H2O
?
show the reaction diagram
Nepenthes mixta
-
preferential cleavage: -Asp, Asp-, Lys-, Ala-
-
-
-
additional information
?
-
-
the enzyme is involved in digestion of prey, mainly insects, for nitrogen uptake of the carnivorous plant, and is therefore secreted into the pitcher fluid
-
-
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
nepenthesin I and II show a high cysteine content which form disulfide bonds, molecular modeling
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
CoSO4
Nepenthes dormaniana, Nepenthes mixta, Nepenthes neufvilleana, Nepenthes sp.
-
-
CuCl2
Nepenthes dormaniana, Nepenthes mixta, Nepenthes neufvilleana, Nepenthes sp.
-
-
cupric ions
-
strong inhibition of nepenthesin I and II by diazoacetyl-DL-norleucine in presence of cupric ions, no inactivation in absence of cupric ions
diazoacetyl-DL-norleucine
-
strong inhibition of nepenthesin I and II in presence of cupric ions, no inactivation in absence of cupric ions
Diazoacetyl-DL-norleucine methyl ester
-
i.e. DAN, strong inhibition of nepenthesin I and II only in presence of Cu2+
Diazoacetyl-DL-norleucine methyl ester
-
i.e. DAN
dithiothreitol
-
isoforms nethepsin I, IIa and IIb are completely inactivated in the presence of 40-70 mM dithiothreitol
DL-Norleucine methyl ester
-
-
HgCl2
Nepenthes dormaniana, Nepenthes mixta, Nepenthes neufvilleana, Nepenthes sp.
-
-
N-(diazoacetyl)-N-(2,4-dinitrophenyl)ethylenediamine
-
-
Pepstatin
-
complete inhibition of nepenthesin I and II under acidic conditions at 0.1 mM
pepstatin A
-
0.1 mM, complete inhibition of nepenthesin I and II
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
2.2
Nepenthes dormaniana, Nepenthes mixta, Nepenthes neufvilleana
-
-
2.6
-
reaction with acid-denatured haemoglobin, nepenthesin I and II
2.6
-
optimum of nepenthesin I and II
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
1.7 - 3.2
-
-
1.7 - 6
Nepenthes dormaniana, Nepenthes mixta, Nepenthes neufvilleana
-
-
additional information
-
pH profile, nepenthesin I and II
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
37
-
assay at
40
Nepenthes sp.
-
assay at
45
-
nepenthesin II
50
Nepenthes dormaniana, Nepenthes mixta, Nepenthes neufvilleana
-
pH 2.2
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
-
additional information
-
temperature profile, nepenthesin I and II
pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
3.09
-
nepenthesin II, calculation from nucleotide sequence
3.94
-
nepenthesin Ia and Ib, calculation from nucleotide sequence
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
Nepenthes sp.
-
-
Manually annotated by BRENDA team
Nepenthes dormaniana, Nepenthes neufvilleana, Nepenthes mixta
-
-
Manually annotated by BRENDA team
-
secretion from
Manually annotated by BRENDA team
-
secretory gland inside the pitcher
Manually annotated by BRENDA team
-
secretory gland inside the pitcher
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
-
the enzyme is secreted to the pitcher fluid
-
Manually annotated by BRENDA team
-
the enzyme is secreted to the pitcher fluid
-
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
45000
-
nepenthesin II, gel filtration
649776
51000
-
nepenthesin I, gel filtration
649776
58000
-
purified nepenthesin I, gel filtration
667380
600000
-
multicomplex structured enzyme
667380
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
monomer
-
1 * 45000
monomer
-
1 * 35000, nepenthesin II, SDS-PSGE under non-reducing conditions, 1 * 37476, nepenthesin Ia, calculation from nucleotide sequence, 1 * 37511, nepenthesin II, calculation from nucleotide sequence, 1 * 37519, nepenthesin Ib, calculation from nucleotide sequence, 1 * 45000, nepenthesin I, SDS-PSGE under non-reducing conditions
monomer
-
1 * 45000, reducing SDS-PAGE, nepenthesin I, 1 * 51000, non-reducing SDS-PAGE, nepenthesisn I, 1 * 35000, reducing SDS-PAGE, nepenthesin II, 1 * 45000, non-reducing SDS-PAGE, nepenthesisn II
additional information
-
primary and tertiary structure determination and analysis and molecular phylogeny, molecular modeling, nepenthesin I and II, overview
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
glycoprotein
-
nepenthesin I contains 6 potential N-glycosylation sites, nepenthesin II has none
glycoprotein
-
nepenthesin I
no glycoprotein
-
nepenthesin II
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
2.9
-
at pH 2.9 at 37C, activity decreased very slowly on longer incubation, about 60% and 30% of the original activity are retained after 30 days and 65 days, respectively
717394
3 - 10
-
30 days, nepenthesin I and II, stable, overview
667380
3 - 10
-
when nepenthesin I is incubated at pH 3.0 at 60C for 30 days, about 30% of the original activity remains. When incubated at pH 3.0-10.0 at 37C for 30 days, the enzyme retains 80% or more of activity
717394
3 - 9
-
40C, highest stability at pH 3.0-9.0
36773
5
-
60C, highest stability at pH 5
36773
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
37
-
pH 3.0, nepenthesin I loses 4% of its activity after 7 days, nepenthesin II loses 10% of its activity after 7 d
649776
37
-
nepenthesin I and II, completely stable for at least 30 days
667380
40
-
pH 3.0-9.0
36773
50
-
pH 3.0, nepenthesin I loses 40% of its activity after 30 d, nepenthesin II loses 56% of its activity after 30 d
649776
50
-
nepenthesin I and II, loss of 40% of activity after 7 days
667380
53
-
stable up to 53C during 1 h of incubation
717394
60
-
highest stability at pH 5
36773
70
-
highly reduced activity of nepenthesin I and II after 7 days
667380
80
-
nepenthesin I and II, complete loss of activity after 7 days
667380
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dithiothreitol
-
isoforms nethepsin I, IIa and IIb are completely inactivated in the presence of 40-70 mM dithiothreitol
guanidine-HCl
-
completely inactivated in the presence of 3 M, the midpoints of denaturation are 4.3 M, 0.4 M and 1.2 M guanidine-HCl for isoform nethepsin I, IIa and IIb, respectively
urea
-
activity is completely lost in the presence of 5 M urea, the midpoints of denaturation are 1.7 M, 2.4 M and 1.6 M urea for isoform nethepsin I, IIa and IIb, respectively
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DEAE-cellulose column chromatography, pepstatin column chromatography, MonoQ column chromatography, and Sephacryl S-200 gel filtration
-
by gel filtration, ion exchange chromatography, and pepstatin affinity chromatography, native enzyme to homogeneity
-
DEAE-cellulose column chromatography, pepstatin column chromatography, MonoQ column chromatography, and Sephacryl S-200 gel filtration
-
native nepenthesin I and II 200fold and 186fold, respectively, from pitcher fluid to homogeneity by two steps of anion exchange chromatography, gel filtration, pepstatin affinity chromatography, and another different step of exchange chromatography
-
nepenthesin I and II
-
-
Nepenthes dormaniana, Nepenthes mixta, Nepenthes neufvilleana
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
nepenthesin I and II
-
nepenthesin I and II, DNA and amino acid sequence determination and analysis, phylogenetic analysis
-