Information on EC 3.4.21.B55 - pyrolysin

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The expected taxonomic range for this enzyme is: Pyrococcus furiosus

EC NUMBER
COMMENTARY hide
3.4.21.B55
preliminary BRENDA-supplied EC number
RECOMMENDED NAME
GeneOntology No.
pyrolysin
-
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
endopeptidase with two preferential cleavage sites in alphaS1-casein, at bonds Gln9-/-Gly10 and Leu16-/-Asn17
show the reaction diagram
-
-
-
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
azocasein + H2O
?
show the reaction diagram
beta-casein + H2O
?
show the reaction diagram
-
-
-
-
?
casein + H2O
?
show the reaction diagram
-
-
-
-
?
D-Val-Leu-Lys-4-nitroanilide + H2O
D-Val-Leu-Lys + 4-nitroanilide
show the reaction diagram
-
41% of the activity compared to succinyl-Ala-Ala-Pro-Lys-4-nitroanilide
-
-
?
kappa-casein + H2O
?
show the reaction diagram
-
-
-
-
?
methoxysuccinyl-Arg-Pro-Tyr-4-nitroanilide + H2O
methoxysuccinyl-Arg-Pro-Tyr + 4-nitroaniline
show the reaction diagram
-
52% of the activity compared to succinyl-Ala-Ala-Pro-Lys-4-nitroanilide
-
-
?
RPKHPIKHQGLPQEVLNENLLRF + H2O
RPKHPIKHQ + GLPQEVL + NENLLRF
show the reaction diagram
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i.e. alphaS1-casein-(1–23) can be completely degraded by pyrolysin resulting in the appearance of four new peptides that are identified based on their retention time. Prolonged incubation shows a decrease of the peptides 1–16 and 10–23 and the appearance of new cleavage products. Pyrolysin is an endopeptidase with two preferential cleavage sites in alphaS1-casein, at bonds 9–10 and 16–17
-
-
?
succinyl-Ala-Ala-Pro-Arg-4-nitroanilide + H2O
succinyl-Ala-Ala-Pro-Arg + 4-nitroaniline
show the reaction diagram
-
32% of the activity compared to succinyl-Ala-Ala-Pro-Lys-4-nitroanilide
-
-
?
succinyl-Ala-Ala-Pro-Lys-4-nitroanilide + H2O
succinyl-Ala-Ala-Pro-Lys + 4-nitroaniline
show the reaction diagram
succinyl-Ala-Ala-Pro-Met-4-nitroanilide + H2O
succinyl-Ala-Ala-Pro-Met + 4-nitroaniline
show the reaction diagram
-
13% of the activity compared to succinyl-Ala-Ala-Pro-Lys-4-nitroanilide
-
-
?
succinyl-Ala-Ala-Pro-Phe-4-nitroanilide + H2O
succinyl-Ala-Ala-Pro-Phe + 4-nitroaniline
show the reaction diagram
-
14% of the activity compared to succinyl-Ala-Ala-Pro-Lys-4-nitroanilide
-
-
?
succinyl-Glu-Ala-Pro-Phe-4-nitroanilide + H2O
succinyl-Glu-Ala-Pro-Phe + 4-nitroaniline
show the reaction diagram
-
7% of the activity compared to succinyl-Ala-Ala-Pro-Lys-4-nitroanilide
-
-
?
succinyl-Lys-Ala-Pro-Phe-4-nitroanilide + H2O
succinyl-Lys-Ala-Pro-Phe + 4-nitroaniline
show the reaction diagram
-
65% of the activity compared to succinyl-Ala-Ala-Pro-Lys-4-nitroanilide
-
-
?
additional information
?
-
-
no activity with Ala-Ala-Phe-4-nitroanilide or pyroglutamyl-Pro-Val-4-nitroanilide
-
-
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mg2+
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supplementation of Na+, Ca2+, or Mg2+ salts at concentrations similar to those in seawater leads to an increase in enzyme activity. The activity level increases with the increase of the salt concentrations from 5 mM to 50 mM
Na+
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supplementation of Na+, Ca2+, or Mg2+ salts at concentrations similar to those in seawater leads to an increase in enzyme activity. The activity level increases with the increase of the salt concentrations from 40 mM to 600 mM
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
diisopropyl fluorophosphate
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0.5 mM, complete inhibition
EDTA
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10 mM, 27% inhibition
PMSF
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1 mM, 87% inhibition
additional information
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no inhibition by cysteine-type protease inhibitors pCMB and iodoacetate
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3.32 - 3.83
succinyl-Ala-Ala-Pro-Lys-4-nitroanilide
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1893 - 3128
succinyl-Ala-Ala-Pro-Lys-4-nitroanilide
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
494 - 942
succinyl-Ala-Ala-Pro-Lys-4-nitroanilide
19752
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 10.5
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broad pH range with at least 50% of its maximal activity between pH 6.5 and 10.5
7.8
-
assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
90
-
assay at
110
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wild-type enzyme and mutant enzyme D818N/D820N
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
90 - 120
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90°C: about 60% of maximal activity, 120°C: about 70% of maximal activity
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
-
75% of the activity is associated with the cell-envelope
Manually annotated by BRENDA team
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25% of the activity was located in the cytoplasm
Manually annotated by BRENDA team
additional information
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no activity is observed in the medium when Pyrococcus furiosus is grown in batch culture
-
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
80000
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x * 80000, low-molecular weight form, SDS-PAGE
105000
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x * 105000, high-molecular weight form, SDS-PAGE
150000
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x * 150000, SDS-PAGE, after acid denaturation, high molecular weight pyrolysin
155000
-
x * 155000, calculated from sequence of pyrolysin precursor, high molecular weight pyrolysin
additional information
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two proteolytically active fractions designated as high and low molecular weight pyrolysin
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
proteolytic modification
-
the maturation of the enzyme involves autoprocessing of both the N-propeptide and a C-terminal propeptide. The long C-terminal extension can be dissected into a C-terminal propeptide and a C-terminal extension (CTEm) that is attached to the catalytic domain of the mature enzyme. Both of these propeptides assist in achieving pyrolysin hyperthermostability
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
80
-
t1/2 is above 6000 min
115
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5 min, wild-type enzyme loses about 75% of its activity, mutant enzyme D818N/D820N loses about 70% of its activity
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
binding of Ca2+ increases the stability of the PPC domain and, in turn, stabilizes the global structure of the enzyme by modulating interdomain interactions
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glycoprotein
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supplementation of Na+, Ca2+, or Mg2+ salts at concentrations similar to those in seawater destabilizes recombinant pyrolysin. Ca2+ and Mg2+ are stronger denaturants than Na+ for pyrolysin. Ca2+ also acts a stabilizer of pyrolysin by binding to the enzyme at specific sites. The destabilizing effect of metal ions on pyrolysin appears to be related to the disturbance of surface electrostatic interactions of the enzyme
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OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
oxygen-stable enzyme
-
722300
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli. At high temperatures, pyrolysin proform (Pls) is converted to mature pyrolysin (mPls) via autoprocessing of both the N- and C-terminal propeptides
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the recombinant proform of Pyrococcus furiosus pyrolysin and several N- and C-terminal deletion mutants are successfully expressed in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D55A
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after teatment at 95°C for 12 h the mutant enzyme displays similar levels of residual activity to that of the wild-type enzyme. When incubated with 2 mM EDTA at 95°C for 10 min, the mutant enzyme is completely inactivated, whereas the wild-type enzyme displays a residual activity of 52.6%
D55A/D58A
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mutant enzyme is capable of maturation at 95°C and also exhibits thermal stability similar to wild-type enzyme in the absence of chelating agents
D55A/D58A/E59A
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the proform of the mutant enzyme is unable to mature at 95°C
D55A/E59A
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less stable than wild-type enzyme under non-chelating conditions
D55N/D58N/E59Q
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less stable than wild-type enzyme under non-chelating conditions
D58A
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after teatment at 95°C for 12 h the mutant enzyme displays similar levels of residual activity to that of the wild-type enzyme. When incubated with 2 mM EDTA at 95°C for 10 min, the mutant enzyme retains 9.6% of its original activity, whereas the wild-type enzyme displays a residual activity of 52.6%
D58A/E59A
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less stable than wild-type enzyme under non-chelating conditions
D818N/D820N
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half-life of the mutant enzyme at 95°C is 18 h, compared to 12 h measured for the wild-type enzyme. Inactivation of the enzymes at 100-115°C results in retention of a higher level of its original activity by the mutant enzyme compared to wild-type enzyme. The mutant enzyme displays higher specific activity towards azocasein than wild-type enzyme in the temperature range of 50-110°C. The mutant enzyme also shows higher initial velocity of hydrolysis than wild-type enzyme with azocasein or succinyl-Ala-Ala-Pro-Lys-4-nitroanilide as substrate
E59A
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after teatment at 95°C for 12 h the mutant enzyme exhibits a slightly lower residual activity than wild-type enzyme. When incubated with 2 mM EDTA at 95°C for 10 min, the mutant enzyme is completely inactivated, whereas the wild-type enzyme displays a residual activity of 52.6%
R276A
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after teatment at 95°C for 12 h the mutant enzyme displays similar levels of residual activity to that of the wild-type enzyme. When incubated with 2 mM EDTA at 95°C for 10 min, the mutant enzyme retains 9.6% of its original activity, whereas the wild-type enzyme displays a residual activity of 52.6%