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Information on EC 3.4.21.B55 - pyrolysin
for references in articles please use BRENDA:EC3.4.21.B55
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preliminary BRENDA-supplied EC number
EC Tree 3 Hydrolases
3.4 Acting on peptide bonds (peptidases)
3.4.21 Serine endopeptidases
3.4.21.B55 pyrolysin
The expected taxonomic range for this enzyme is: Pyrococcus furiosus
- 3.4.21.B55
-
hyperthermophilic
-
pyrococcus
- furiosus
-
subtilisin-like
-
subtilases
- archaeon
-
hyperthermostable
-
n-glycosylation
- asn
-
autoproteolysis
- subtilisin
Reaction Schemes
showendopeptidase with two preferential cleavage sites in alphaS1-casein, at bonds Gln9-/-Gly10 and Leu16-/-Asn17
Synonyms
pyrolysin, more
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REACTION 
REACTION DIAGRAM
COMMENTARY 
ORGANISM
UNIPROT
LITERATURE
endopeptidase with two preferential cleavage sites in alphaS1-casein, at bonds Gln9-/-Gly10 and Leu16-/-Asn17
-
-
-
-
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
LITERATURE
COMMENTARY
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
D-Val-Leu-Lys-4-nitroanilide + H2O
D-Val-Leu-Lys + 4-nitroaniline
Substrates: 41% of the activity compared to succinyl-Ala-Ala-Pro-Lys-4-nitroanilide
Products: -
Products: -
?
methoxysuccinyl-Arg-Pro-Tyr-4-nitroanilide + H2O
methoxysuccinyl-Arg-Pro-Tyr + 4-nitroaniline
Substrates: 52% of the activity compared to succinyl-Ala-Ala-Pro-Lys-4-nitroanilide
Products: -
Products: -
?
RPKHPIKHQGLPQEVLNENLLRF + H2O
RPKHPIKHQ + GLPQEVL + NENLLRF
Substrates: i.e. alphaS1-casein-(123) 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 116 and 1023 and the appearance of new cleavage products. Pyrolysin is an endopeptidase with two preferential cleavage sites in alphaS1-casein, at bonds 910 and 1617
Products: -
Products: -
?
succinyl-AAPK-4-nitroanilide + H2O
succinyl-AAPK + 4-nitroaniline
-
Substrates: -
Products: -
Products: -
?
succinyl-Ala-Ala-Pro-Arg-4-nitroanilide + H2O
succinyl-Ala-Ala-Pro-Arg + 4-nitroaniline
Substrates: 32% of the activity compared to succinyl-Ala-Ala-Pro-Lys-4-nitroanilide
Products: -
Products: -
?
succinyl-Ala-Ala-Pro-Met-4-nitroanilide + H2O
succinyl-Ala-Ala-Pro-Met + 4-nitroaniline
Substrates: 13% of the activity compared to succinyl-Ala-Ala-Pro-Lys-4-nitroanilide
Products: -
Products: -
?
succinyl-Ala-Ala-Pro-Phe-4-nitroanilide + H2O
succinyl-Ala-Ala-Pro-Phe + 4-nitroaniline
Substrates: 14% of the activity compared to succinyl-Ala-Ala-Pro-Lys-4-nitroanilide
Products: -
Products: -
?
succinyl-Glu-Ala-Pro-Phe-4-nitroanilide + H2O
succinyl-Glu-Ala-Pro-Phe + 4-nitroaniline
Substrates: 7% of the activity compared to succinyl-Ala-Ala-Pro-Lys-4-nitroanilide
Products: -
Products: -
?
succinyl-Lys-Ala-Pro-Phe-4-nitroanilide + H2O
succinyl-Lys-Ala-Pro-Phe + 4-nitroaniline
Substrates: 65% of the activity compared to succinyl-Ala-Ala-Pro-Lys-4-nitroanilide
Products: -
Products: -
?
succinyl-Ala-Ala-Pro-Lys-4-nitroanilide + H2O
succinyl-Ala-Ala-Pro-Lys + 4-nitroaniline
Substrates: -
Products: -
Products: -
?
succinyl-Ala-Ala-Pro-Lys-4-nitroanilide + H2O
succinyl-Ala-Ala-Pro-Lys + 4-nitroaniline
-
Substrates: -
Products: -
Products: -
?
additional information
?
-
Substrates: no activity with Ala-Ala-Phe-4-nitroanilide or pyroglutamyl-Pro-Val-4-nitroanilide
Products: -
Products: -
?
additional information
?
-
-
Substrates: no activity with Ala-Ala-Phe-4-nitroanilide or pyroglutamyl-Pro-Val-4-nitroanilide
Products: -
Products: -
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Ca2+
Mg2+
-
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+
-
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
Ca2+
-
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 0.1 mM to 10 mM
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
no inhibition by cysteine-type protease inhibitors pCMB and iodoacetate
-
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
3.32
succinyl-Ala-Ala-Pro-Lys-4-nitroanilide
pH 7.5, 90°C, mature enzyme
3.83
succinyl-Ala-Ala-Pro-Lys-4-nitroanilide
pH 7.5, 90°C, mature enzyme lacking the C-terminal extension (mPlsDELTAC740)
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1893
succinyl-Ala-Ala-Pro-Lys-4-nitroanilide
pH 7.5, 90°C, mature enzyme lacking the C-terminal extension (mPlsDELTAC740)
3128
succinyl-Ala-Ala-Pro-Lys-4-nitroanilide
pH 7.5, 90°C, mature enzyme
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
494
succinyl-Ala-Ala-Pro-Lys-4-nitroanilide
pH 7.5, 90°C, mature enzyme lacking the C-terminal extension (mPlsDELTAC740)
942
succinyl-Ala-Ala-Pro-Lys-4-nitroanilide
pH 7.5, 90°C, mature enzyme
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
6.5 - 10.5
broad pH range with at least 50% of its maximal activity between pH 6.5 and 10.5
7.5
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
95
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
90 - 120
-
90°C: about 60% of maximal activity, 120°C: about 70% of maximal activity
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
75% of the activity is associated with the cell-envelope
brenda
25% of the activity was located in the cytoplasm
brenda
additional information
no activity is observed in the medium when Pyrococcus furiosus is grown in batch culture
-
brenda
Highest Expressing Human Cell Lines
Cell Line LinksPlease wait a moment until the data is sorted. This message will disappear when the data is sorted.
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). PLS_PYRFU
Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1)
1398
0
154475
Swiss-Prot
-
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
150000
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
additional information
two proteolytically active fractions designated as high and low molecular weight pyrolysin
additional information
-
two proteolytically active fractions designated as high and low molecular weight pyrolysin
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
?
x * 150000, SDS-PAGE, after acid denaturation, high molecular weight pyrolysin
?
x * 155000, calculated from sequence of pyrolysin precursor, high molecular weight pyrolysin
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POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY
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
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D132A
-
the purified mature form of the variant enzyme exhibits specific activity similar to that of wild-type enzyme. The heat inactivation profile of the mutant enzyme is similar to that of the wild-type enzyme at 95°C. It retains lower residual activities than that of the wild-type enzyme following incubation with EGTA at 95°C. These data indicate that the mutated residue is involved in calcium binding at the Ca3 site, which is required to stabilize pyrolysin
D132A/D136A
-
the purified mature form of the variant enzyme exhibits specific activity similar to that of wild-type enzyme
D132A/N134A
-
the purified mature form of the variant enzyme exhibits specific activity similar to that of wild-type enzyme. In the presence of EGTA, the protein quantity of very unstable variants enzyme does not decrease as much as that of the wild-type enzyme following heat treatment at 95°C
D132A/N134A/D136A
-
the purified mature form of the variant enzyme exhibits specific activity similar to that of wild-type enzyme
D132N/D136N
-
the heat inactivation profile of the mutant enzyme is similar to that of the wild-type enzyme at 95°C. It retains lower residual activities than that of the wild-type enzyme following incubation with EGTA at 95°C. These data indicate that the mutated residue is involved in calcium binding at the Ca3 site, which is required to stabilize pyrolysin
D136A
-
the purified mature form of the variant enzyme exhibits specific activity similar to that of wild-type enzyme. The heat inactivation profile of the mutant enzyme is similar to that of the wild-type enzyme at 95°C. It retains lower residual activities than that of the wild-type enzyme following incubation with EGTA at 95°C. These data indicate that the mutated residue is involved in calcium binding at the Ca3 site, which is required to stabilize pyrolysin
D138A/Q139A/E140A/D141A
-
the mutant enzyme displays heat resistance similar to that of the WT under nonchelating conditions but exhibits decreased residual activity compared to the wild-type enzyme following incubation with EGTA at 95°C. These results suggest that the residues downstream of motif 1 also contribute to calcium binding at the Ca3 site
D161A/D163A
-
the variant proform is capable of maturation, but the yield of mature enzyme is very low
D161A/D163A/D165A/S441A
-
the D161A/D163A/D165A/S441A proform degrades more than the the S441A proform at 95°C, confirming that the replacement of Asp161, Asp163, and Asp165 with Ala affects structural stability of the proform and makes it more susceptible to thermogenic hydrolysis
D161N/D163N/D165N
-
the variant proform is capable of maturation, but the yield of mature enzyme is very low. Heat inactivation profile at 95° is similar to wild-type enzyme, either in the absence or presence of EGTA. Similar activity levels as cpompared to wild-type enzyme
D163A
-
the variant proform is capable of maturation, but the yield of mature enzyme is very low. Heat inactivation profile at 95° is similar to wild-type enzyme, either in the absence or presence of EGTA. Slightly lower activity levels as cpompared to wild-type enzyme
D163A/D165A
-
the variant proform is capable of maturation, but the yield of mature enzyme is very low
D165A
-
the variant proform is capable of maturation, but the yield of mature enzyme is very low
D55A
-
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
-
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
-
the proform of the mutant enzyme is unable to mature at 95°C
D55A/E59A
-
less stable than wild-type enzyme under non-chelating conditions
D55N/D58N/E59Q
-
less stable than wild-type enzyme under non-chelating conditions
D58A
-
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
-
less stable than wild-type enzyme under non-chelating conditions
D818N/D820N
E59A
-
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%
F135R
-
the thermostability of the variant does not change under nonchelating conditions compared to that of the wild-type. The variant shows a lower level of residual activity than the wild-type enzyme following incubation with EGTA at 95°C. This result implies that a positively charged Arg residue in Dx[DN]xDG motif 1 affects calcium binding at the Ca3 site
N134A
-
the purified mature form of the variant enzyme exhibits specific activity similar to that of wild-type enzyme. The heat inactivation profile of the mutant enzyme is similar to that of the wild-type enzyme at 95°C. It retains lower residual activities than that of the wild-type enzyme following incubation with EGTA at 95°C. These data indicate that the mutated residue is involved in calcium binding at the Ca3 site, which is required to stabilize pyrolysin
N134A/D136A
-
the purified mature form of the variant enzyme exhibits specific activity similar to that of wild-type enzyme. In the presence of EGTA, the protein quantity of very unstable variants enzyme does not decrease as much as that of the wild-type enzyme following heat treatment at 95°C
R249E
-
the mutation within insert IS29 improves enzyme thermostability and resistance to salt-induced autocleavage. This is likely due to removing unfavorable electrostatic interactions involving Arg249. Half-life at 95°C is 18 h compared to 12 h for wild-type enzyme
R249E/D818N/D820N
-
the mutant enzyme is both resistant to salt induced autocleavage and exhibits a remarkably long half-life of approximately 36 h at 95°C. Half-life at 95°C is 36 h compared to 12 h for wild-type enzyme
R276A
-
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%
D818N/D820N
-
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
D818N/D820N
-
half-life at 95°C is 18 h compared to 12 h for wild-type enzyme
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
100
105
110
115
-
5 min, wild-type enzyme loses about 75% of its activity, mutant enzyme D818N/D820N loses about 70% of its activity
95
additional information
-
extra structural elements play a crucial role in adapting pyrolysin to hyperthermal environments
105
-
20 min, wild-type enzyme loses about 45% of its activity, mutant enzyme D818N/D820N loses about 30% of its activity
110
-
5 min, wild-type enzyme loses about 30% of its activity, mutant enzyme D818N/D820N loses about 20% of its activity
95
-
half-life of wild-type enzyme: 12 h, half-life of mutant enzyme D818N/D820N is 18 h
95
-
half-life: 12 h (wild-type enzyme), 18 h (mutant enzymes R249E and D818N/D820N), 36 h (R249E/D818N/D820N)
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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
-
four inserts in the catalytic domain of hyperthermostable pyrolysin contribute to the folding, maturation, stability, and activity of the enzyme at high temperatures. The modification of extra structural elements in pyrolysin-like proteases is a promising strategy for modulating global structure stability and enzymatic activity of this class of protease
-
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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ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SDS
-
mature insert-deletion variants are characterized and show that insert IS29 and insert IS8 contribute to enzyme activity and stability, respectively. In the presence of NaCl, pyrolysin undergoes autocleavage at two sites. One within insert IS29 and the other in insert IS27. Disrupting the ion pairs in IS27 and IS8 induces autocleavage in the absence of salts. Autocleavage products combine noncovalently to form an active, nicked enzyme that is resistant to SDS and urea denaturation
urea
-
mature insert-deletion variants are characterized and show that insert IS29 and insert IS8 contribute to enzyme activity and stability, respectively. In the presence of NaCl, pyrolysin undergoes autocleavage at two sites. One within insert IS29 and the other in insert IS27. Disrupting the ion pairs in IS27 and IS8 induces autocleavage in the absence of salts. Autocleavage products combine noncovalently to form an active, nicked enzyme that is resistant to SDS and urea denaturation
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OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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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
-
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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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Dai, Z.; Fu, H.; Zhang, Y.; Zeng, J.; Tang, B.; Tang, X.F.
Insights into the maturation of hyperthermophilic pyrolysin and the roles of its N-terminal propeptide and long C-terminal extension
Appl. Environ. Microbiol.
78
4233-4241
2012
Pyrococcus furiosus (P72186), Pyrococcus furiosus
brenda
Eggen, R.; Geerling, A.; Watts, J.; de Vos,W.M.
Characterization of pyrolysin, a hyperthermoactive serine protease from the archaebacterium Pyrococcus furiosus
FEMS Microbiol. Lett.
71
17-20
1990
Pyrococcus furiosus (P72186)
-
brenda
Voorhorst, W.G.; Eggen, R.I.; Geerling, A.C.; Platteeuw, C.; Siezen, R.J.; Vos, W.M.
Isolation and characterization of the hyperthermostable serine protease, pyrolysin, and its gene from the hyperthermophilic archaeon Pyrococcus furiosus
J. Biol. Chem.
271
20426-20431
1996
Pyrococcus furiosus (P72186), Pyrococcus furiosus
brenda
de Vos, W.M.; Voorhorst, W.G.; Dijkgraaf, M.; Kluskens, L.D.; van der Oost, J.; Siezen, R.J.
Purification, characterization, and molecular modeling of pyrolysin and other extracellular thermostable serine proteases from hyperthermophilic microorganisms
Methods Enzymol.
330
383-393
2001
Pyrococcus furiosus (P72186)
brenda
Zeng, J.; Gao, X.; Dai, Z.; Tang, B.; Tang, X.F.
Effects of metal ions on stability and activity of hyperthermophilic pyrolysin and further stabilization of this enzyme by modification of a Ca2+-binding site
Appl. Environ. Microbiol.
80
2763-2772
2014
Pyrococcus furiosus
brenda
Gao, X.; Zeng, J.; Yi, H.; Zhang, F.; Tang, B.; Tang, X.F.
Four inserts within the catalytic domain confer extra stability and activity to hyperthermostable pyrolysin from Pyrococcus furiosus
Appl. Environ. Microbiol.
83
e03228-16
2017
Pyrococcus furiosus
brenda
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