Information on EC 3.4.23.1 - pepsin A

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

EC NUMBER
COMMENTARY hide
3.4.23.1
-
RECOMMENDED NAME
GeneOntology No.
pepsin A
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Preferential cleavage: hydrophobic, preferably aromatic, residues in P1 and P1' positions. Cleaves Phe1-/-Val, Gln4-/-His, Glu13-/-Ala, Ala14-/-Leu, Leu15-/-Tyr, Tyr16-/-Leu, Gly23-/-Phe, Phe24-/-Phe and Phe25-/-Tyr bonds in the B chain of insulin
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of peptide bond
-
-
endopeptidase
-
CAS REGISTRY NUMBER
COMMENTARY hide
9001-75-6
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
duck
-
-
Manually annotated by BRENDA team
kid
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
dogfish
-
-
-
Manually annotated by BRENDA team
pepsinogen isoform A1; Japanese seabass
UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
japanese monkey
-
-
Manually annotated by BRENDA team
rice field eel
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
palometa
-
-
Manually annotated by BRENDA team
turtle
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
salmon
-
-
-
Manually annotated by BRENDA team
shark
-
-
-
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
acetyl-Ala-Ala-Lys-Phe(4-NO2)-Ala-Ala-NH2 + H2O
?
show the reaction diagram
-
-
-
-
?
acetyl-L-phenylalanyl-L-diiodotyrosine + H2O
acetyl-L-phenylalanine + L-diiodotyrosine
show the reaction diagram
AFPLEFEREL + H2O
AFPLEF + EREL
show the reaction diagram
AFPLEFFREL + H2O
AFPLEF + FREL
show the reaction diagram
AFPLEFIREL + H2O
AFPLEF + IREL
show the reaction diagram
benzyloxycarbonyl-Gly-Ala-(4-nitro)Phe-Trp-NHCH2CH2OH + H2O
?
show the reaction diagram
-
-
-
-
?
benzyloxycarbonyl-His-(4-nitro)Phe-Phe-OMet + H2O
?
show the reaction diagram
-
-
-
-
?
benzyloxycarbonyl-His-Phe(NO2)-beta-phenyl-L-lactyl-O-methyl ester + H2O
benzyloxycarbonyl-His-Phe(NO2) + phenyl-L-lactyl-O-methyl ester
show the reaction diagram
benzyloxycarbonyl-His-Phe-Phe-NH2 + H2O
?
show the reaction diagram
-
-
-
-
?
benzyloxycarbonyl-His-Phe-Phe-O-methyl ester + H2O
?
show the reaction diagram
-
-
-
-
?
benzyloxycarbonyl-His-Phe-Trp-OEt + H2O
?
show the reaction diagram
-
-
-
-
?
benzyloxycarbonyl-His-Tyr-Phe-O-methyl ester + H2O
?
show the reaction diagram
-
-
-
-
?
bis-phenyl sulfite + H2O
?
show the reaction diagram
-
-
-
-
?
casein + H2O
?
show the reaction diagram
Cry1A(b) protein + H2O
?
show the reaction diagram
-
from transgenic maize or from Bacillus thuringiensis
-
-
?
dynorphin A (1-7) + H2O
?
show the reaction diagram
Hemoglobin + H2O
?
show the reaction diagram
HPHLSFMAI + H2O
HPHLSF + Met-Ala-Ile + ?
show the reaction diagram
insulin + H2O
?
show the reaction diagram
KPAEFF(NO2)-AL + H2O
?
show the reaction diagram
-
-
-
-
?
KPAEFF(NO2)AL + H2O
?
show the reaction diagram
KPAEFFRL + H2O
KPAEF + FRL
show the reaction diagram
KPIEF(NO2)RL + H2O
?
show the reaction diagram
-
-
-
?
KPIKF(NO2)RL + H2O
?
show the reaction diagram
-
-
-
?
KPILF(NO2)RL + H2O
?
show the reaction diagram
-
-
-
?
KPIQF(NO2)RL + H2O
?
show the reaction diagram
-
-
-
?
KPPEF(NO2)RL + H2O
?
show the reaction diagram
-
-
-
?
KYSSWYVAL + H2O
KYSSW + YVAL
show the reaction diagram
L-Lys-L-Pro-L-Ala-L-Glu-L-Phe-L-Phe(NO2)-L-Ala-L-Leu + H2O
?
show the reaction diagram
-
-
-
-
?
Leu-Ser-Phe(NO2)-Nle-Ala-Leu + H2O
?
show the reaction diagram
-
-
-
-
?
Leu-Ser-Phe(p-NO2)-Nle-Ala-Leu + H2O
?
show the reaction diagram
-
-
-
?
LSF(NO2)-Nle-AL + H2O
?
show the reaction diagram
-
-
-
?
Lys-Pro-Ala-Glu-Phe-Phe(4NO2)-Ala-Leu + H2O
?
show the reaction diagram
Lys-Pro-Ala-Glu-Phe-Phe(NO2)-Ala-Leu + H2O
?
show the reaction diagram
-
-
-
-
?
Lys-Pro-Ala-Glu-Phe-Phe(p-NO2)-Ala-Leu + H2O
?
show the reaction diagram
-
-
-
?
Mca-KKPAEFFALK-Dnp + H2O
Mca-KKPAEF + FFALK-Dnp
show the reaction diagram
this peptide is preferentially cleaved at Phe-Phe, with a second minor cleavage occurring at Phe-Ala
-
-
?
Mca-KLHPEVLFVLEK-Dnp + H2O
Mca-KLHPEVL + FVLEK-Dnp
show the reaction diagram
the preferred cleavage site is between Leu-Phe, a minor cleavage site is between Phe-Ala
-
-
?
metmyoglobin + H2O
?
show the reaction diagram
-
-
treatment with pepsin at pH 4.0 results in lowering the (pseudo)peroxidase activity of metmyoglobin both at physiological pH and at meat pH, leading to strongly enhanced prooxidative effect of mildly proteolyzed metmyoglobin on lipid oxidation
-
?
MOCAc-Ala-Pro-Ala-Lys-Phe-Phe-Arg-Leu-Lys(Dnp)-NH2 + H2O
?
show the reaction diagram
-
-
-
-
?
N,N-dimethyl-casein + H2O
?
show the reaction diagram
N,N-dimethylhemoglobin + H2O
?
show the reaction diagram
-
-
-
-
?
N-trifluoroacetyl aromatic L-amino acids + H2O
?
show the reaction diagram
-
-
-
-
?
NT/NMN (142-151) + H2O
KIPYIL + KRQL
show the reaction diagram
ovalbumin + H2O
?
show the reaction diagram
-
-
-
-
?
oxidized insulin B chain + H2O
FVNQHLCGSHLVEAL + L-Tyr + LVCGERGFFYTPKA
show the reaction diagram
two major cleavage sites are at Leu15-Tyr16 and Tyr16-Leu17
-
-
?
Oxidized insulin B-chain + H2O
?
show the reaction diagram
-
-
-
?
Phe-Gly-His-(4-nitro)Phe-Phe-Ala-Phe-OMe + H2O
?
show the reaction diagram
-
-
-
-
?
POMC (165-174) + H2O
AFPLE + FKREL
show the reaction diagram
Pro-Thr-Glu-Lys-Phe(4-NO2)-Arg-Leu-NH2 + H2O
?
show the reaction diagram
-
-
-
-
?
Pro-Thr-Glu-Phe-(4-nitro)Phe-Arg-Leu + H2O
?
show the reaction diagram
-
-
-
-
?
Pro-Thr-Glu-Phe-(p-nitro-Phe)-Arg-Leu-OH + H2O
Pro-Thr-Glu-Phe + (p-nitro-Phe)-Arg-Leu-OH
show the reaction diagram
-
-
-
?
Pro-Thr-Glu-Phe-Phe(4-NO2)-Arg-Leu + H2O
?
show the reaction diagram
-
-
-
-
?
Proteins + H2O
?
show the reaction diagram
Ribonuclease + H2O
?
show the reaction diagram
-
reduced and carboxymethylated ribonuclease A
-
?
serum albumin + H2O
?
show the reaction diagram
dogfish
-
-
-
-
?
SGGYDLSFLPQPPQE + H2O
?
show the reaction diagram
-
predominant cleavage of the peptide corresponding to the carboxy-terminal telopeptide region of bovine type I collagen alpha1 chain at Asp-Leu, Leu-Ser and Phe-Leu and at a significant lower rate at Ser-Phe
-
?
substance P + H2O
RPKPQQF + FGLM
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
Proteins + H2O
?
show the reaction diagram
additional information
?
-
E2IKQ9
the cleavage site of isoform PG1 to produce pepsin is clearly at the 41-42 bond of Phe-Ala
-
-
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
CaCl2
-
5 mM, 1.39fold activation
CoCl2
-
5 mM, 1.34fold activation
MgCl2
-
10 mM, 1.24fold activation
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,2-epoxy-3-(4-nitrophenoxy)propane
alpha2-Macroglobulin
-
activity towards reduced and carboxymethylated ribonuclease A is significantly inhibited at pH 5.5, the activity towards a peptide substrate, oxidized insulin B-chain, is scarcely inhibited
-
amastatin
97.2 residual activity at 0.01 mM
amylopectin sulfate
-
bestatin
92.7 residual activity at 0.01 mM
Bis-(beta-chloroethyl)sulfide
Diazoacetyl-DL-norleucine methyl ester
dimethyl sulfoxide
-
1%, complete loss of activity
dithiothreitol
E-64
89.5 residual activity at 0.01 mM
imidazole
iodoacetamide
82.1 residual activity at 0.05 mM
leupeptin
91.9 residual activity at 0.01 mM
N-bromosuccinimide
NaCl
-
30%, activity of pepsin A decreases by 60-65%
p-bromophenylacyl bromide
pefabloc
87.8 residual activity at 1 mM
Pepstatin
pepstatin A
peptides
PI-3
-
inhibitor from Ascaris suum
-
poly-L-lysine
prosegment K36A-mutant of pepsinogen
-
N-terminal, 44 residue prosegment
-
prosegment of pepsinogen
-
N-terminal, 44 residue prosegment
-
prosegment R8A-mutant of pepsinogen
-
N-terminal, 44 residue prosegment
-
prosegment V4A-mutant of pepsinogen
-
N-terminal, 44 residue prosegment
-
Tetranitromethane
Val-D-Leu-Pro-Phe-Phe-Val-D-Leu
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.16
acetyl-Ala-Ala-Lys-Phe(4-NO2)-Ala-Ala-NH2
-
T77D/G78(S)S79 double mutant
-
0.3 - 0.4
AFPLEFEREL
0.084 - 0.17
AFPLEFFREL
0.079 - 0.098
AFPLEFIREL
0.49
benzyloxycarbonyl-His-Phe-Phe-NH2
-
-
0.18
benzyloxycarbonyl-His-Phe-Phe-O-ethyl ester
-
-
0.37
benzyloxycarbonyl-His-Phe-Phe-O-methyl ester
-
-
0.25
benzyloxycarbonyl-His-Trp-Phe-O-methyl ester
-
-
0.68
benzyloxycarbonyl-His-Tyr-Phe-O-methyl ester
-
-
0.76 - 1.05
casein
0.055 - 0.12
hemoglobin
-
0.034 - 0.083
KPAEFF(NO2)AL
0.011 - 0.035
KPIEF(NO2)RL
0.03 - 0.16
KPIKF(NO2)RL
0.0051 - 0.0093
KPILF(NO2)RL
0.015 - 0.05
KPIQF(NO2)RL
0.013 - 0.074
KPPEF(NO2)RL
0.048 - 0.075
L-Lys-L-Pro-L-Ala-L-Glu-L-Phe-L-Phe(NO2)-L-Ala-L-Leu
0.019
Leu-Ser-Phe(NO2)-Nle-Ala-Leu
-
-
0.011 - 0.044
Leu-Ser-Phe(p-NO2)-Nle-Ala-Leu
0.015 - 0.041
LSF(NO2)-Nle-AL
0.034
Lys-Pro-Ala-Glu-Phe-Phe(4NO2)-Ala-Leu
-
-
0.0433
Lys-Pro-Ala-Glu-Phe-Phe(NO2)-Ala-Leu
-
pH 5.3, 25C
0.043 - 0.098
Lys-Pro-Ala-Glu-Phe-Phe(p-NO2)-Ala-Leu
0.0054
Mca-KKPAEFFALK-Dnp
at pH 4.0 and 37C
0.05
N,N-dimethylcasein
-
-
-
0.046
N,N-dimethylhemoglobin
-
-
-
0.022 - 0.029
POMC (165-174)
0.15 - 0.18
Pro-Thr-Glu-Lys-Phe(4-NO2)-Arg-Leu-NH2
0.025 - 0.074
Pro-Thr-Glu-Phe-(p-nitro-Phe)-Arg-Leu-OH
0.04 - 0.2
Pro-Thr-Glu-Phe-Phe(4-NO2)-Arg-Leu
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
8.9 - 45
AFPLEFEREL
85 - 472
AFPLEFFREL
56 - 203
AFPLEFIREL
0.21
benzyloxycarbonyl-His-Phe-Phe-NH2
Sus scrofa
-
-
0.31
benzyloxycarbonyl-His-Phe-Phe-O-ethyl ester
Sus scrofa
-
-
0.15
benzyloxycarbonyl-His-Phe-Phe-O-methyl ester
Sus scrofa
-
-
0.013
benzyloxycarbonyl-His-Trp-Phe-O-methyl ester
Sus scrofa
-
-
0.013
benzyloxycarbonyl-His-Tyr-Phe-O-methyl ester
Sus scrofa
-
-
7.34 - 50
hemoglobin
-
5 - 96
KPAEFF(NO2)AL
0.24 - 97.8
KPIEF(NO2)RL
0.63 - 55.5
KPIKF(NO2)RL
0.8 - 60.4
KPILF(NO2)RL
0.1 - 97.7
KPIQF(NO2)RL
0.21 - 79.8
KPPEF(NO2)RL
16 - 162
L-Lys-L-Pro-L-Ala-L-Glu-L-Phe-L-Phe(NO2)-L-Ala-L-Leu
1.2 - 88.7
Leu-Ser-Phe(p-NO2)-Nle-Ala-Leu
56
Lys-Pro-Ala-Glu-Phe-Phe(NO2)-Ala-Leu
Sus scrofa
-
pH 5.3, 25C
2.5 - 181
Lys-Pro-Ala-Glu-Phe-Phe(p-NO2)-Ala-Leu
0.03
Mca-KKPAEFFALK-Dnp
Shewanella amazonensis
A1S3N8
at pH 4.0 and 37C
7
N,N-dimethylcasein
Ovis aries
-
-
-
18.3
N,N-dimethylhemoglobin
Ovis aries
-
-
-
2.7 - 3.7
POMC (165-174)
0.54 - 3.32
Pro-Thr-Glu-Phe-(p-nitro-Phe)-Arg-Leu-OH
additional information
additional information
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
29 - 110
AFPLEFEREL
12112
1000 - 2800
AFPLEFFREL
12114
730 - 2100
AFPLEFIREL
12113
130 - 190
hemoglobin
1730
2000
Lys-Pro-Ala-Glu-Phe-Phe(NO2)-Ala-Leu
Sus scrofa
-
pH not specified in the publication, temperature not specified in the publication
41332
5.6
Mca-KKPAEFFALK-Dnp
Shewanella amazonensis
A1S3N8
at pH 4.0 and 37C
41956
93 - 170
POMC (165-174)
19620
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0000000457
pepstatin A
-
at 25C, pH not specified in the publication
0.0000956
prosegment K36A-mutant of pepsinogen
-
pH 5.3, 25C
-
0.0000362
prosegment of pepsinogen
-
pH 5.3, 25C
-
0.0000583
prosegment R8A-mutant of pepsinogen
-
pH 5.3, 25C
-
0.0000824
prosegment V4A-mutant of pepsinogen
-
pH 5.3, 25C
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
1.8
-
N,N-dimethylcasein, N,N-dimethylhemoglobin
1.85
-
N-acetyl-L-phenylalanyl-L-diiodotyrosine
2.1
-
-
2.2
-
-
4.6
-
benzyloxycarbonyl-L-histidyl-L-phenylalanyl-L-tryptophan ethyl ester
additional information
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.5 - 3.5
-
N,N-dimethylhemoglobin, pH 0.5: about 30% of maximum activity, pH 3.5: about 15% of maximum activity
0.5 - 5
-
N,N-dimethylcasein, pH 0.5: about 35% of maximum activity, pH 5.0: about 20% of maximum activity
1 - 3.5
1 - 4
pH 1.0: about 70% of maximal activity, pH 4.0: about 50% of maximal activity
1.5 - 4
-
pH 1.5: about 75% of maximum activity, pH 4.0: about 30% of maximum activity
1.5 - 4.5
1.6 - 2.2
-
-
2.5 - 4
-
pH 2.5: about 55% of maximal activity, pH 4.0: about 55% of maximal activity
3.5 - 5
50% activity is retained at pH 5.0 At pH 6.0, the enzyme shows no activity
additional information
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40
-
the activity of pepsin isoform 1 is largely lost at higher than 50C
42 - 50
maximum activity is observed at temperatures between 42 and 50C, decreasing so drastically above 50C that complete loss of activity is detected at 60C
50 - 65
-
-
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 55
-
20C: about 40% of maximal activity, 55C: about 60% of maximal activity
25 - 50
25C: about 70% of maximal activity, 50C: about 90% of maximal activity
25 - 60
25C: about 50% of maximal activity, 60C: about 10% of maximal activity
30 - 80
-
-
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3
-
or below, isoelectric focusing
5.1
-
pepsinogen 1, isoelectric focusing
5.3
pepsinogen isoform PG1, isoelectric focusing
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
abomasal tissue
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30000
x * 30000, pepsin isoform P1, SDS-PAGE
34250
ESI mass spectrometry
40000
-
gel filtration
50000
x * 50000, SDS-PAGE
additional information
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
-
1 * 35000, SDS-PAGE
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phosphoprotein
-
contains 0.37 molecules phosphorous per molecule
proteolytic modification
side-chain modification
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystallization in presence of dimethyl sulfoxide. One dimethyl sulfoxide molecule occupies a site on the surface of enzyme interacting with two of its residues. In crystal structure, conformation of enzyme remains unchanged. In solution, dimethyl sulfoxide causes a slight change in secondary structure and complete loss of activity
-
hanging-drop vapour diffusion method, crystal structure of the complex between pepsin inhibitor-3 and the enzyme at 1.75 A and at 2.45 A resolution. In the enzyme-inhibitor complex, the N-terminal beta-strand of pepsin inhibitor-3 pairs with one strand of the active site flap (residues 70-82) of pepsin, thus forming an eight-stranded beta-sheet that spans the two proteins
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
1 - 4.5
-
24 h, 20C
36758
2.2 - 5.9
-
45C, pH 2.16: 7% loss of activity after 40 min, pH 5.93: 12% loss of activity after 40 min
36733
3 - 10
-
pepsin A is completely inactivated at pH 3.0-10.0 at 37C after 30 days, the enzyme is unstable at pH 7.0 and above (irreversible inhibition)
717394
6
-
room temperature, 30 min, stable below
679335
6 - 6.5
-
maximum stability
36759
7.5
-
rapid denaturation
36758
8.5
-
stable up to
36755
10
-
30 min, about 80% loss of activity
36748
additional information
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
38.8
-
melting point of wild-type. For glycosylated mutants, melting point increases by 5-17 degrees
60
-
15 min, about 80% loss of activity
additional information
-
-
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
1,4-dioxane
-
incubation for 20 min at pH 2 and 25C, the enzyme retains its activity in aqueous 1,4-dioxane with increasing organic solvent concentration up to 30%
acetonitrile
-
incubation for 20 min at pH 2 and 25C, the enzyme retains its activity in aqueous acetonitrile with increasing organic solvent concentration up to 60%
Ethanol
-
incubation for 20 min at pH 2 and 25C, the enzyme retains its activity in aqueous ethanol with increasing organic solvent concentration up to 60%
guanidine-HCl
-
pepsin A is stable up to 3.6 M and inactivated at 5 M guanidine-HCl
Methanol
-
aqueous methanol cryosolvents: no deleterious effect
urea
-
pepsin A is stable up to at least 5.6 M urea
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, dialyzed
-
-20C, pH 4.4, 2-3 months
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
affinity chromatography of pepsin and pepsinogen using immobilized ligands derived from the specific substrate for this enzyme, optimum conditions of an interaction of the enzyme and its zymogen with immobilized ligands derived from the enzyme substrate: N-acetyl-L-Phe-3,5-diiodo-L-tyrosine
-
ammonium sulfate precipitation, DEAE-Sephacel column chromatography, and Sephacryl S-200 gel filtration
ammonium sulfate precipitation, DEAE-Sephacel gel filtration, and Sephacryl S-200 gel filtration
-
binding to immobilized inhibitor Val-D-Leu-Pro-Phe-Phe-Val-D-Leu
-
expressed in Escherichia coli
-
from rennet
-
HisTrap column chromatography and Superdex 200 gel filtration
multiple isoforms
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stomach extract
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) cells
expression in Escherichia coli
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expression in Escherichia coli CJ236
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overexpression in Escherichia coli; overexpression in Escherichia coli
wild-type, D32E, D215E, and D32E/D215E thioredoxin-pepsinogen fusion proteins are expressed in Escherichia coli GI724
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
L291S
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replacement of a pepsin A-specific residue by a chymosin-specific one. Decrease in hydrolysis of peptides with hydrophobic/aromatic residues at P1 site, increase in hydrolysis of peptides with charged residues at P1
L291S/L298Q
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replacement of a pepsin A-specific residue by a chymosin-specific one. Decrease in hydrolysis of peptides with hydrophobic/aromatic residues at P1 site, increase in hydrolysis of peptides with charged residues at P1
L298Q
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replacement of a pepsin A-specific residue by a chymosin-specific one. Decrease in hydrolysis of peptides with hydrophobic/aromatic residues at P1 site, increase in hydrolysis of peptides with charged residues at P1
M289D
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replacement of a pepsin A-specific residue by a chymosin-specific one. Decrease in hydrolysis of peptides with hydrophobic/aromatic residues at P1 site, increase in hydrolysis of peptides with charged residues at P1
D37A
the mutant is completely inactive towards the fluorogenic substrate Mca-KKPAEFFALK-Dnp at pH 4.0
D215E
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unlike wild-type, the mutant fusion protein is incapable of autocatalytic activation upon acidification of the medium, as determined by Western blot analysis. Mature mutant pepsin is obtained by processing fusion protein samples through an immobilized pepsin column. Tm-value of mutant enzyme is 65C compared to 71C for wild-type enzyme. The pH activity profiles of wild-type and mutant pepsin is similar. Mutant enzyme has a stronger affinity for the synthetic substrate KPAEFF(NO2)AL. Turnover number for mutant enzyme is significantly lower than that of the wild-type. kcat/Km is 1.9fold lower than wild-type value
D32E
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unlike wild-type, the mutant fusion protein is incapable of autocatalytic activation upon acidification of the medium, as determined by Western blot analysis. Mature mutant pepsin is obtained by processing fusion protein samples through an immobilized pepsin column. Tm-value of mutant enzyme is 63C compared to 71C for wild-type enzyme. Pronounced decrease in activity below pH 2.5. Mutant enzyme has a stronger affinity for the synthetic substrate KPAEFF(NO2)AL. Turnover number for mutant enzyme is significantly lower than that of the wild-type. kcat/Km is 8.7fold lower than wild-type value
D32E/D215E
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unlike wild-type, the mutant fusion protein is incapable of autocatalytic activation upon acidification of the medium, as determined by Western blot analysis. Mature mutant pepsin is obtained by processing fusion protein samples through an immobilized pepsin column. Tm-value of mutant enzyme is 63C compared to 71C for wild-type enzyme. The pH activity profiles of wild-type and mutant pepsin is similar. KM-value for KPAEFF(NO2)AL is not significantly different relative to wild-type. Turnover number for mutant enzyme is significantly lower than that of the wild-type. kcat/Km is fold lower than wild-type value. kcat/Km is 13.3fold lower than wild-type value
DELTA240-246/+GD
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inactivation rate at pH 7.0 is 86% of that of the wild-type value. The Km-value for KPAEFF(NO2)AL is 2fold higher than that of the wild-type enzyme. The KM-value for LSF(NO2)-Nle-AL as substrate is 79% of the wild-type value
E244N/V246T
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introduction of N-glycosylation site. Glycosylated mutant shows similar KM-value and substrate specificity as wild-type, but reduced catalytic efficiency
E287M
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the ratio of turnover number to KM-value for KPILF(NO2)RL is 2.2fold higher than that of the wild-type enzyme, the ratio of turnover number to KM-value for KPIEF(NO2)RL is 1.3fold higher than that of the wild-type enzyme,the ratio of turnover number to KM-value for KPIQF(NO2)RL is 1.6fold higher than that of the wild-type enzyme, the ratio of turnover number to KM-value for KPIKF(NO2)RL is 60% of that of the wild-type enzyme, the ratio of turnover number to KM-value for KPPEF(NO2)RL is 90% of that of the wild-type enzyme
F111T/L112F
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the ratio of turnover number to KM-value for KPILF(NO2)RL is 3.8fold higher than that of the wild-type enzyme, the ratio of turnover number to KM-value for KPIEF(NO2)RL is 1.9fold higher than that of the wild-type enzyme,the ratio of turnover number to KM-value for KPIQF(NO2)RL is 2.3fold higher than that of the wild-type enzyme, the ratio of turnover number to KM-value for KPIKF(NO2)RL is 1.7fold higher than that of the wild-type enzyme, the ratio of turnover number to KM-value for KPPEF(NO2)RL is 2.9fold higher than that of the wild-type enzyme. Mutant enzyme cleaves SGGYDLSFLPQPPQE at one site Leu-Ser, compared to three sites cleaved by the wild-type enzyme, and at a rate 23fold higher than that of the wild-type enzyme
F111T/L112F/E287M
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the ratio of turnover number to KM-value for KPILF(NO2)RL is 5.8fold higher than that of the wild-type enzyme, the ratio of turnover number to KM-value for KPIEF(NO2)RL is 2.8fold higher than that of the wild-type enzyme,the ratio of turnover number to KM-value for KPIQF(NO2)RL is 2.8fold higher than that of the wild-type enzyme, the ratio of turnover number to KM-value for KPIKF(NO2)RL is 1.1fold higher than that of the wild-type enzyme, the ratio of turnover number to KM-value for KPPEF(NO2)RL is 3.7fold higher than that of the wild-type enzyme
F111T/L112F/T222V/E287M
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the ratio of turnover number to KM-value for KPILF(NO2)RL is 3.5fold higher than that of the wild-type enzyme, the ratio of turnover number to KM-value for KPIEF(NO2)RL is 2.4fold higher than that of the wild-type enzyme,the ratio of turnover number to KM-value for KPIQF(NO2)RL is 4.4fold higher than that of the wild-type enzyme, the ratio of turnover number to KM-value for KPIKF(NO2)RL is 2.2fold higher than that of the wild-type enzyme, the ratio of turnover number to KM-value for KPPEF(NO2)RL is 5.1fold higher than that of the wild-type enzyme
G2C/L167C
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the mutation reduces the inactivation rate at pH 7.0 by 1.8times compared to wild-type value. The Km-value for KPAEFF(NO2)AL is 2.35fold higher than that of the wild-type enzyme. The KM-value for LSF(NO2)-Nle-AL as substrate is 95% of the wild-type value
G2S/D3Y
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the mutation reduces the inactivation rate at pH 7.0 by 1.4times compared to wild-type value. The Km-value for KPAEFF(NO2)AL is 2.2fold higher than that of the wild-type enzyme. The KM-value for LSF(NO2)-Nle-AL as substrate is 1.5fold higher than that of the wild-type enzyme
G2S/D3Y/L10M/T12A/E13S
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the mutation reduces the inactivation rate at pH 7.0 by 5.8times. In presence of glycerol and sucrose, this mutant shows a very low rate of inactivation, the residual activity after 240 min is 50% of the initial activity compared to the wild-type which loses most of its activity in 60 min. The Km-value for KPAEFF(NO2)AL is 2.4fold higher than that of the wild-type enzyme. The KM-value for LSF(NO2)-Nle-AL as substrate is 1.5fold higher than that of the wild-type enzyme
G76A
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mutant enzyme with lower catalytic efficiency and is activated more slowly than the wild-type enzyme. The slower activation process is associated directly with altered proteolytic activity
G76S
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mutant enzyme with lower catalytic efficiency and is activated more slowly than the wild-type enzyme. The slower activation process is associated directly with altered proteolytic activity
G76V
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mutant enzyme with lower catalytic efficiency and is activated more slowly than the wild-type enzyme. The slower activation process is associated directly with altered proteolytic activity
G78(S)S79
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differences in substrate specificity and catalytic activity
L10M/T12A/E13S
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the mutation reduces the inactivation rate at pH 7.0 by 1.5times compared to wild-type value. The Km-value for KPAEFF(NO2)AL is 1.9fold higher than that of the wild-type enzyme. The KM-value for LSF(NO2)-Nle-AL as substrate is 2.2fold higher than that of the wild-type enzyme
S110N/L112T
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introduction of N-glycosylation site. Glycosylated mutant shows similar KM-value and substrate specificity as wild-type, but reduced catalytic efficiency
S196R/D200G/E202K
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inactivation rate at pH 7.0 is 86% of that of the wild-type value
S281N
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introduction of N-glycosylation site. Glycosylated mutant shows similar KM-value and substrate specificity as wild-type, but reduced catalytic efficiency
S46K/D52N/N54K/Q55R/D60K
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inactivation rate at pH 7.0 is 91% of that of the wild-type value
S46K/D52N/N54K/Q55R/D60K/S196R/D200G/E202K
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the mutation reduces the inactivation rate at pH 7.0 by 2.3times compared to wild-type value. Km-value for KPAEFF(NO2)AL is 1.7fold higher than that of the wild-type enzyme. The KM-value for LSF(NO2)-Nle-AL as substrate is 1.6fold higher than that of the wild-type enzyme
T222V
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the ratio of turnover number to KM-value for KPILF(NO2)RL is 2.5fold higher than that of the wild-type enzyme, the ratio of turnover number to KM-value for KPIEF(NO2)RL is 1.1fold higher than that of the wild-type enzyme,the ratio of turnover number to KM-value for KPIQF(NO2)RL is 1.4fold higher than that of the wild-type enzyme, the ratio of turnover number to KM-value for KPIKF(NO2)RL is equal to that of the wild-type enzyme, the ratio of turnover number to KM-value for KPPEF(NO2)RL is 1.9fold higher than that of the wild-type enzyme
T222V/E287M
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the ratio of turnover number to KM-value for KPILF(NO2)RL is 4.1fold higher than that of the wild-type enzyme, the ratio of turnover number to KM-value for KPIEF(NO2)RL is 2.6fold higher than that of the wild-type enzyme,the ratio of turnover number to KM-value for KPIQF(NO2)RL is 2.1fold higher than that of the wild-type enzyme, the ratio of turnover number to KM-value for KPIKF(NO2)RL is 80% of that of the wild-type enzyme, the ratio of turnover number to KM-value for KPPEF(NO2)RL is 2.9fold higher than that of the wild-type enzyme. Mutant enzyme cleaves site Ser-Phe in SGGYDLSFLPQPPQE at a rate 20fold higher than the wild-type enzyme
T77D
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differences in substrate specificity and catalytic activity
T77D/G78(S)S79
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double mutant, differences in substrate specificity and catalytic activity
T77N
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introduction of N-glycosylation site. Glycosylated mutant shows similar KM-value and substrate specificity as wild-type, but reduced catalytic efficiency
additional information
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two chain mutant
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
acid refolded pepsin has secondary and tertiary structures intermediate between the alkaline denatured and native forms but is thermodynamically stable relative to the native state. The acid refolded state of pepsin is dependent on the protein concentration during refolding. Both the secondary and tertiary structures of concentrated-refolded pepsin are native-like, in contrast to the intermediate nature of acid refolded pepsin, refolded under dilute concentration. Despite a native-like conformation, concentrated-refolded pepsin is more stable and has substantially reduced activity compared to that of the native state, suggesting that the protein is misfolded. It is proposed that the stable but misfolded, acid-refolded states are evidence that pepsin in its native conformation is metastable
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alkaline denatured pepsin
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renaturation of the supernatant containing pepsinogen A1 is achieved by a 200fold dilution of the denaturant in 100 mm Tris/HCl buffer (pH 8.0); renaturation of the supernatant containing pepsinogen A2 is achieved by a 200fold dilution of the denaturant in 100 mm Tris/HCl buffer (pH 8.0)
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
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combined use of a theoretical model that relates electrophoretic behaviour of peptides to their sequence together with capillary electrophoresis-mass spectrometry to characterize the cleavage specificity of recombinant enzymes. Characterization of a protein lysate using recombinant and natural pepsin
food industry
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treatment with pepsin at pH 4.0 results in lowering the (pseudo)peroxidase activity of metmyoglobin both at physiological pH and at meat pH, leading to strongly enhanced prooxidative effect of mildly proteolyzed metmyoglobin on lipid oxidation
medicine
nutrition
synthesis
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comparison of hydrolysis kinetics of hemoglobin after immobilization of enzyme on aluminium oxide and on 2-ethanolamine-O-phosphate-modified acidic alumina. Modified alumina results in comparatively less adsorption of peptides and complete adsorption of heme
Show AA Sequence (111 entries)
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