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Information on EC 3.4.21.B57 - pernisine and Organism(s) Thermococcus kodakarensis and UniProt Accession P58502
for references in articles please use BRENDA:EC3.4.21.B57preliminary BRENDA-supplied EC number
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3.4.21.B57 pernisineSpecify your search results
Word Map
- 3.4.21.B57
-
hyperthermophilic
- archaeon
-
subtilisin-like
- pernix
-
aeropyrum
- subtilisins
-
thermococcus
- prion
- medicine
- kodakaraensis
-
proregion
-
mesophilic
-
autoprocessed
- detergents
- edta
-
codon-optimised
-
far-uv
- tk-subtilisin
-
high-temperature
- cacl2
- n-propeptide
-
roll
-
hyperthermostable
The taxonomic range for the selected organisms is: Thermococcus kodakarensis
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Reaction Schemes
the enzyme can digest the pathological prion protein isoform (PrPSc) from different species, e.g. human, bovine, deer and mouse
Synonyms
pernisine, tk-sp, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
Tk-subtilisin
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
N-succinyl-AAPF-4-nitroanilide + H2O
N-succinyl-AAPF + 4-nitroaniline
-
-
-
?
N-succinyl-Ala-Ala-Pro-Leu-4-nitroanilide + H2O
N-succinyl-Ala-Ala-Pro-Leu + 4-nitroaniline
24.3% compared to the activity with N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide
-
-
?
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide + H2O
N-succinyl-Ala-Ala-Pro-Phe + 4-nitroaniline
-
-
-
?
pro-Tk-subtilisin + H2O
Tk-subtilisin + propeptide
autoactivation
-
-
?
Tk-RNase H2 + H2O
?
ribonuclease H2 from Thermococcus kodakarensis, pulse proteolysis using the superstable subtilisin-like serine protease Tk-subtilisin in highly concentrated guanidine hydrochloride to unfold the highly stable substrate protein. The native state of Tk-RNase H2 is completely resistant to Tk-subtilisin, whereas the unfolded state (induced by 4 M GdnHCl) is degraded by Tk-subtilisin, identification of the cleavage sites. Structure analysis of unfolded substrate states
-
-
?
abnormal prion protein PrP(Sc) + H2O
?
prion protein PrP(Sc) is a pathological prion protein PrP isoform. The enzyme can disrupt PrPSc to a level undetectable by Western-blot analysis
-
-
?
abnormal prion protein PrP(Sc) + H2O
?
the abnormal prion protein (scrapie-associated prion protein, PrP(Sc)) is considered to be included in the group of infectious agents of transmissible spongiform encephalopathies. Although PrP(Sc) is known to be resistant toward proteolytic enzymes, Tk-subtilisin is able to degrade PrP(Sc) under extreme conditions
-
-
?
additional information
?
-
broad substrate specificity, with a slight preference for aromatic or large nonpolar P1 substrate residues
-
-
?
additional information
?
-
the propeptide is effectively degraded by the mature enzyme only at high temperatures, because it is too stable to be degraded at moderate temperatures
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
pro-Tk-subtilisin + H2O
Tk-subtilisin + propeptide
autoactivation
-
-
?
additional information
?
-
the propeptide is effectively degraded by the mature enzyme only at high temperatures, because it is too stable to be degraded at moderate temperatures
-
-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Ca2+
CaCl2
highest activity in the presence of 5 mM CaCl2. The enzyme exhibits 70% and 80% of the maximal activity in the presence of 1 and 100 mM CaCl2, respectively
Ca2+
the enzyme contains seven Ca2+ ions. Four of them (Ca2-Ca5) are responsible for folding of Tk-subtilisin. Ca6 and Ca7 ions are not important for activity. The Ca1 ion is required for the maximal activity of Tk-subtilisin. Ca1, Ca6, and Ca7 ions, especially the Ca1 ion, contribute to the hyperthermostabilization of Tk-subtilisin
Ca2+
the enzyme requires Ca2+ for folding and assumes a molten globule-like structure in the absence of Ca2+ even in the presence of Tk-propeptide. Tk-subtilisin contains seven Ca2+-binding sites. Four of them are located within a long loop, which mostly consists of a unique insertion sequence of this protein
Ca2+
bound at the high-affinity Ca1 site and low-affinity Ca2 site
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
AMPHITOL 20Y-B
presence of 1% (w/v) AMPHITOL 20Y-B causes strong inhibitions, resulting in an enzyme retaining only 50% of its activity
Thermococcus kodakaraensis serpin
irreversibly inhibits more strongly at 80°C than at 40°C. The covalent inhibitory complex is highly stable and the ester bond between serpin and protease can be hydrolyzed only in a harsh condition, in which most proteases are denatured
-
Thermococcus kodakaraensis subtilisin propeptide
potent noncompetitive inhibitor of the mature domain
-
additional information
the activity is retained or even enhanced in the presence of nonionic, cationic (except in the presence of SANIZOL C), and amphoteric surfactants at both 0.1 and 1% (w/v)
-
EDTA
about 20% loss of activity in the presence of 0.01% (w/v) at 80°C, about 60% loss of activity in the presence of 1% (w/v) at 80°C
EDTA
activity decreases with the increasing concentration of EDTA from 0.01 to 1% (w/v)
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
destabilization of the hydrophobic core of Tk-propeptide by a nonpolar-to-polar amino acid substitution is an effective way to increase the activation rate of Pro-Tk-subtilisin
-
additional information
the activities in the presence of 1% SANISOL C and AMPHITOL 20 N are higher than those at the concentration of 0.1%. This may relate to the critical micelle concentration of the surfactants. Improvement of the enzyme activity in the presence of nonionic surfactants might be due to the stimulation of conformational changes in Tk-SP or the substrate, leading to activity gain
-
additional information
-
the activities in the presence of 1% SANISOL C and AMPHITOL 20 N are higher than those at the concentration of 0.1%. This may relate to the critical micelle concentration of the surfactants. Improvement of the enzyme activity in the presence of nonionic surfactants might be due to the stimulation of conformational changes in Tk-SP or the substrate, leading to activity gain
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
8
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide
80°C, pH 9.5, the Km value of 8.0 mM represents the apparent value, because the solubility of the substrate at pH 9.5 is too low to increase its concentration beyond 5 mM
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.000025
Thermococcus kodakaraensis subtilisin propeptide
20°C, pH 9.5
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.5
9.5
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
80
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
40 - 90
the enzyme exhibits 10% to 20% of the maximal activity at 40°C or 90°C
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
the enzyme is secreted into the external medium, probably as inactive pro-enzyme, with the assistance of the signal sequence
-
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
prepro-Tk-subtilisin (Prepro-TKS), which consists of the signal sequence [Met (-24)-Ala(-1)], propeptide (Tkpro, Gly1-Leu69), and mature domain (Tk-subtilisin, Gly70-Gly398). the pro-enzyme form contains the insertion sequence, IS1, at the N-terminus of the mature domain which is required not only for hyperstabilization of Pro-TKS but also for its rapid maturation
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
44000
63000
gel filtration, Pro-Tk-S359C (an enzyme derivative with the mutation of the active-site serine residue to Cys)
66000
x * 66000, Pro-Tk-S359C (an enzyme derivative with the mutation of the active-site serine residue to Cys)
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
?
x * 66000, Pro-Tk-S359C (an enzyme derivative with the mutation of the active-site serine residue to Cys)
monomer
monomer
1 * 45000, active-site mutant S255A of pro-Tk-subtilisin, SDS-PAGE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
proteolytic modification
proteolytic modification
pro-subtilisin is inactive in the absence of Ca2+ but is activated upon autoprocessing and degradation of propeptide in the presence of Ca2+ at 80°C. This maturation process is completed within 30 min at 80°C but is bound at an intermediate stage, in which the propeptide is autoprocessed from the mature domain (mat-subtilisin) but forms an inactive complex with mat-subtilisin*, at lower temperatures. At 80°C, approximately 30% of the pro-subtilisin is autoprocessed into propeptide and mat-subtilisin, and the other 70% is completely degraded to small fragments. mat-Subtilisin is inactive in the absence of Ca2+ but is activated upon incubation with Ca2+ at 80°C
proteolytic modification
produced from its inactive precursor, Pro-Tk-subtilisin (Gly1-Gly398), by autoprocessing and degradation of the propeptide (Tk-propeptide, Gly1-Leu69). This activation process is extremely slow at moderate temperatures owing to the high stability of Tk-propeptide. The refolding rate of Pro-F17H/S324A and autoprocessing rate of Pro-F17H/S324C are nearly identical to those of their parent proteins (Pro-S324A and Pro-S324C). The activation rate of Pro-F17H greatly increases when compared with that of Pro-Tk-subtilisin, such that Pro-F17H is efficiently activated even at 40°C
proteolytic modification
the enzyme is autoprocessed from its precursor with N- and C-propeptides
proteolytic modification
the enzyme matures from the inactive precursor, Pro-Tk-subtilisin (Pro-TKS), upon autoprocessing and degradation of the propeptide (Tkpro)
proteolytic modification
the N-propeptide is autoprocessed first in the maturation process of Pro-Tk-S359C (an enzyme derivative with the mutation of the active-site serine residue to Cys), although the C-propeptide is subsequently autoprocessed and degraded only in the absence of Ca2+. The C-propeptide is not autoprocessed in the presence of Ca2+, suggesting that Pro-Tk-SP derivative lacking N-propeptide (Val114-Gly640) (ProC-Tk-SP) is not an intermediate form but is the mature form of the enzyme. It is shown that the C-propeptide contributes to the stabilization of ProC-Tk-S359C
proteolytic modification
Tk-subtilisin (the mature domain of Pro-Tk-subtilisin in active form (Gly70-Gly398)) is matured from Pro-Tk-subtilisin (pro form (Gly1-Gly398)) upon autoprocessing and degradation of propeptide. Extremely slow maturation at mild temperatures. Maturation rate is greatly increased by a single Gly56/Ser mutation in the propeptide region
proteolytic modification
Tk-subtilisin is matured from Pro-Tk-subtilisin upon autoprocessing and degradation of Tk-propeptide. Tk-subtilisin does not require Tk-propeptide for folding but requires it for acceleration of folding
proteolytic modification
Tk-subtilisin, a subtilisin homologue (Gly70-Gly398) from Thermococcus kodakarensis, is matured from its precursor, Pro-Tk-subtilisin (Tk-subtilisin in a pro form (Gly1-Gly398)), by autoprocessing and degradation of propeptide (Tk-propeptide, a propeptide of Tk-subtilisin (Gly1-Leu69)). The scissile peptide bond between Leu69 and Gly70 of Pro-Tk-subtilisin is first self-cleaved to produce an inactive Tk-propeptide:Tk-subtilisin complex, in which the C-terminal region of Tk-propeptide binds to the active-site cleft of Tk-subtilisin. Tk-propeptide is then dissociated from Tk-subtilisin and degraded by Tk-subtilisin to release active Tk-subtilisin
proteolytic modification
autocatalytic processing, pro-Tk-subtilisin from Thermococcus kodakarensis is fully folded, because it does not require the structural rearrangement upon autoprocessing for the formation of the Ca2+-binding Ca1 site due to the presence of the insertion sequence IS1 between the propeptide and subtilisin domains
proteolytic modification
prepro-Tk-subtilisin (Prepro-TKS), which consists of the signal sequence [Met (-24)-Ala(-1)], propeptide (Gly1-Leu69), and mature domain (Tk-subtilisin, Gly70-Gly398). Tk-subtilisin matures from Pro-Tk-subtilisin upon autoprocessing and degradation of propeptide. The pro-enzyme form contains the insertion sequence, IS1, at the N-terminus of the mature domain which is required not only for hyperstabilization of Pro-Tk-subtilisin but also for its rapid maturation, Most part of IS1 (Gly70-Gly78) is autocatalytically removed when Pro-TKS matures to Tk-subtilisin, structure and mechanism, overview
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
crystallization of the active-site mutant S255A of pro-Tk-subtilisin. The crystal is grown at 4°C by the sitting-drop vapour-diffusion method. Native X-ray diffraction data are collected to 2.3 A resolution.They crystal belongs to the orthorhombic space group I222, with unit-cell parameters a = 92.69, b = 121.78, c = 77.53 A. Assuming the presence of one molecule per asymmetric unit, the Matthews coefficient V(M) was calculated to be 2.6 A(3) Da(-1) and the solvent content was 53.1%
crystallization of the Pro-S324A/DELTACa6 mutant enzyme using the sitting-drop vapor-diffusion method at 4°C
DeltaCa2-Pro-S324A (Ca2+-binding site Ca 2 is removed) is crystallized using sitting-drop vapor-diffusion method at 4°C. DeltaCa3-Pro-S324A (Ca2+-binding site Ca3 is removed) is crystallized using hanging-drop method at 20°C. The structures of DeltaCa2-Pro-S324A (Ca2+-binding site Ca 2 is removed) and DeltaCa3-Pro-S324A (Ca2+-binding site Ca3 is removed) are identical to that of Pro-S324A, except that they lack the Ca2 and Ca3 sites, respectively, and the structure of the Ca2+-binding loop is destabilized. These proteins are slightly more stable than Pro-S324A. These results suggest that the Ca2+-binding loop is required for folding of Tk-subtilisin but does not seriously contribute to the stabilization of Tk-subtilisin in a native structure. The counting of amino acids refers to the enzyme protein without the signal peptide (amino acid 1-24) and the propeptide (amino acid 25-106)
sitting-drop vapour-diffusion method at 4°C. The crystal structure of the active site mutant of Tk-subtilisin (S324A-subtilisin), which is refolded in the presence of Ca2+ and absence of Tk-propeptide, is determined at 2.16 A resolution. This structure is the same as that of Tk-subtilisin matured from Pro-Tk-subtilisin
sitting-drop, vapor-diffusion method at 20 °C, the crystal structure of the active-site mutant of the proenzyme lacking C-propeptide (ProN-Tk-S359A) is determined at 2.0 A resolution
the crystal structure of Pro-F17H/S324A is nearly identical to that of Pro-S324A, indicating that the mutation does not affect the structure of Pro-Tk-subtilisin
the crystal structure of the complex between L69P-propeptide and S324A-subtilisin (i.e. a protease activity-defective mutant) reveals that the C-terminal region of L69P-propeptide does not well fit into the substrate binding pockets of Tk-subtilisin (S1-S4 subsites) as a result of a conformational change caused by the mutation
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Pro-Tk-S359C
construction of an enzyme derivative with the mutation of the active-site serine residue to Cys (Pro-Tk-S359C). Pro-Tk-S359C is purified mostly in an autoprocessed form in which the N-propeptide is autoprocessed but the isolated N-propeptide (ProN) forms a stable complex with ProC-Tk-S359C, indicating that the N-propeptide is autoprocessed first
ProC-Tk-S359C
construction of an enzyme derivative lacking the N-propeptide (ProC-Tk-S359C). The C-propeptide is autoprocessed and degraded when ProC-Tk-S359C is incubated at 80 °C in the absence of Ca2+. However, it is not autoprocessed in the presence of Ca2+. The enzymatic activity of ProC-Tk-S359C is higher than (but comparable to) that of Tk-S359C, an enzyme derivatives lacking both propeptides, suggesting that the C-propeptide is not important for activity. The Tm value of ProC-Tk-S359C is higher than that of Tk-S359C by 25.9°C in the absence of Ca2+ and 7.5 °C in the presence of Ca2+, indicating that the C-propeptide contributes to the stabilization of ProC-Tk-S359C
S324A
S324C
site-directed mutagenesis, structure comparison of the mutant pro-enzyme with the wild-type pro-enzyme
S359C
S359C is more stable than S359A. Tm value of is 58.0°C in the presence of 2.5 M GdnHCl and the absence of Ca2+ and 80.1°C in the presence of 6 m GdnHCl and 10 mm CaCl2
Tk-S359C
construction of an enzyme derivative lacking both propeptides (Tk-S359C). The enzymatic activity of ProC-Tk-S359C, an enzyme derivatives lacking the N-propeptide is higher than (but comparable to) that of Tk-S359C, suggesting that the C-propeptide is not important for activity. The Tm value of ProC-Tk-S359C is higher than that of Tk-S359C by 25.9°C in the absence of Ca2+ and 7.5 °C in the presence of Ca2+, indicating that the C-propeptide contributes to the stabilization of ProC-Tk-S359C
additional information
S324A
the crystal structure of the active site mutant of Tk-subtilisin (S324A-subtilisin), which is refolded in the presence of Ca2+ and absence of Tk-propeptide, is determined at 2.16 A resolution. This structure is the same as that of Tk-subtilisin matured from Pro-Tk-subtilisin. The counting of amino acids refers to the enzyme protein without the signal peptide (amino acid 1-24) and the propeptide (amino acid 25-106)
S324A
site-directed mutagenesis, structure comparison of the mutant pro-enzyme with the wild-type pro-enzyme
additional information
construction of a series of active-site mutants of with (Tk-S359A/C) and without (Tk-S359A/CDeltaJ) beta-jelly roll domain. Both Tk-S359C and Tk-S359CDeltaJ exhibit protease activities, indicating that the beta-jelly roll domain is not required for folding or activity. The Tm value of Tk-S359ADeltaJ determined by far-UV CD spectroscopy in the presence of 10-mM CaCl2 is lower than that of Tk-S359A by 29.4°C. The Tm value of Tk-S359A is decreased by 29.5 °C by the treatment with 10 mM ethylenediaminetetraacetic acid, indicating that the beta-jelly roll domain contributes to the stabilization of Tk-S359A only in a Ca2+-bound form
additional information
-
construction of a series of active-site mutants of with (Tk-S359A/C) and without (Tk-S359A/CDeltaJ) beta-jelly roll domain. Both Tk-S359C and Tk-S359CDeltaJ exhibit protease activities, indicating that the beta-jelly roll domain is not required for folding or activity. The Tm value of Tk-S359ADeltaJ determined by far-UV CD spectroscopy in the presence of 10-mM CaCl2 is lower than that of Tk-S359A by 29.4°C. The Tm value of Tk-S359A is decreased by 29.5 °C by the treatment with 10 mM ethylenediaminetetraacetic acid, indicating that the beta-jelly roll domain contributes to the stabilization of Tk-S359A only in a Ca2+-bound form
additional information
construction of enzyme derivatives with the mutation of the active-site serine residue to Cys (Pro-Tk-S359C), Pro-Tk-S359C derivative lacking the N-propeptide (ProC-Tk-S359C) and both propeptides (Tk-S359C), and a His-tagged form of the isolated C-propeptide (ProC*). Comparison of the susceptibility of ProC* to proteolytic degradation in the presence and absence of Ca2+ suggests that the C-propeptide becomes highly resistant to proteolytic degradation in the presence of Ca2+
additional information
-
construction of enzyme derivatives with the mutation of the active-site serine residue to Cys (Pro-Tk-S359C), Pro-Tk-S359C derivative lacking the N-propeptide (ProC-Tk-S359C) and both propeptides (Tk-S359C), and a His-tagged form of the isolated C-propeptide (ProC*). Comparison of the susceptibility of ProC* to proteolytic degradation in the presence and absence of Ca2+ suggests that the C-propeptide becomes highly resistant to proteolytic degradation in the presence of Ca2+
additional information
Pro-Tk-subtilisin variants with complete amino acid substitutions at Gly56 are constructed. Pro-G56W, Pro-G56E and Pro-G56S are overproduced, purified, and characterized. Their maturation rates increase in the order wild-type enzyme or = G56W-propeptide > G56S-propeptide > G56E-propeptide, indicating that they are inversely correlated with the maturation rates of Pro7-Tk-subtilisin and its derivatives
additional information
the Leu69Pro mutation in the propeptide accelerates the maturation of Pro-Tk-subtilisin by reducing the binding ability of Tk-propeptide to Tk-subtilisin
additional information
the Pro-Tk-subtilisin derivative with the F17His mutation (Pro-F17H), Tk-propeptide derivative with the same mutation (F17H-propeptide), and two active-site mutants of Pro-F17H (Pro-F17H/S324A and Pro-F17H/S324C) are constructed
additional information
to analyze the role of the Ca2+-binding loop, three mutant proteins, Deltaloop-Tk-subtilisin (Ca2+-binding loop is removed), DeltaCa2-Pro-S324A (Ca2+-binding site Ca2 is removed), and DeltaCa3-Pro-S324A (Ca2+-binding site Ca3 is removed), are constructed. The structures of DeltaCa2-Pro-S324A (Ca2+-binding site Ca 2 is removed) and DeltaCa3-Pro-S324A (Ca2+-binding site Ca3 is removed) are identical to that of Pro-S324A, except that they lack the Ca2 and Ca3 sites, respectively, and the structure of the Ca2+-binding loop is destabilized. These proteins are slightly more stable than Pro-S324A
additional information
generation of IS1-deletion mutants of S324A and S324C enzyme variants
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
100
58
Tm-value of Tk-S359C in absence of CaCl2. The Tm value of ProC-Tk-S359C is higher than that of Tk-S359C by 25.9°C in the absence of CaCl2 and 7.5°C in the presence of 10 mM CaCl2, indicating that the C-propeptide of ProC-Tk-S359C contributes to the stabilization of the protein by 25.9°C in Tm in the absence of Ca2+ and 7.5°C in Tm in the presence of Ca2+
58.9
Tm value of a mutant enzyme without beta-jelly roll domain (Tk-S359A/CDeltaJ), 10 mM CaCl2
80
80.1
Tm-value of Tk-S359C in presence of 10 mM CaCl2. The Tm value of ProC-Tk-S359C is higher than that of Tk-S359C by 25.9°C in the absence of CaCl2 and 7.5°C in the presence of 10 mM CaCl2, indicating that the C-propeptide of ProC-Tk-S359C contributes to the stabilization of the protein by 25.9°C in Tm in the absence of Ca2+ and 7.5°C in Tm in the presence of Ca2+
83.9
Tm-value of ProC-Tk-S359C in absence of CaCl2. The Tm value of ProC-Tk-S359C is higher than that of Tk-S359C by 25.9°C in the absence of CaCl2 and 7.5°C in the presence of 10 mM CaCl2, indicating that the C-propeptide of ProC-Tk-S359C contributes to the stabilization of the protein by 25.9°C in Tm in the absence of Ca2+ and 7.5°C in Tm in the presence of Ca2+
87.6
Tm-value of ProC-Tk-S359C in presence of 10 mM CaCl2. The Tm value of ProC-Tk-S359C is higher than that of Tk-S359C by 25.9°C in the absence of CaCl2 and 7.5°C in the presence of 10 mM CaCl2, indicating that the C-propeptide of ProC-Tk-S359C contributes to the stabilization of the protein by 25.9°C in Tm in the absence of Ca2+ and 7.5°C in Tm in the presence of Ca2+
90
additional information
80
half life: more than 60 min, in the presence of 50 mM CaCl2
additional information
attachment of a beta-jelly roll domain to the C-terminus is one of the strategies of the proteins from hyperthermophiles to adapt to high-temperature environment
additional information
-
attachment of a beta-jelly roll domain to the C-terminus is one of the strategies of the proteins from hyperthermophiles to adapt to high-temperature environment
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
in the presence of 0.05% (w/v) nonionic surfactants, such as EMULGEN LS-114 or RHEODOL Tw-0120 V, and 0.01% (w/v) EDTA, Tk-SP retains almost its entire initial activity
in the presence of anionic surfactants, the enzyme is unstable, losing up to 80% of its activity
in the presence of anionic surfactants, Tk-SP is unstable, losing up to 80% of its activity
the activity is retained or even enhanced in the presence of nonionic, cationic (except in the presence of SANIZOL C), and amphoteric surfactants at both 0.1 and 1% (w/v)
the beta-jelly roll domain is required for hyperstabilization of the protein. This domain contains two Ca2+ ions and contributes to the stabilization of the protein only in a Ca2+-bound form
the enzyme is highly stable in the presence of 0.05% (w/v) nonionic surfactants and 0.01% (w/v) EDTA, retaining up to 80% of its activity
the enzyme is highly stable in the presence of both 0.1 and 1% (w/v) nonionic surfactants
the enzyme retains its activity in the presence of surfactants tested at 80°C and 90°C
the enzyme retains more than 100% of its activity in the presence of four of the nonionic surfactants, namely, EMULGEN 147, EMULGEN LS-114, EMULGEN PP-290, and RHEODOL Tw-0120 V. It is less stable in the presence of QUARTAMIN 60 W
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
guanidine-HCl
Tk-subtilisin retains its specific activity in 6 M guanidine HCl after 60 min incubation at room temperature
SDS
Tk-subtilisin shows no significant reduction of specific activity after 60 min incubation at room temperature with 5% SDS
urea
Tk-subtilisin retains its specific activity in 8 M urea after 60 min incubation at room temperature
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
mutant proteins, Deltaloop-Tk-subtilisin (Ca2+-binding loop is removed), DeltaCa2-Pro-S324A (Ca2+-binding site Ca2 is removed), and DeltaCa3-Pro-S324A (Ca2+-binding site Ca3 is removed), are purified in a Ca2+-bound form
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression of wid-type and mutant enzymes in Escherichia coli strain BL21(DE3)
overproduced in Escherichia coli in a form with a putative prosequence in inclusion bodies, solubilized in the presence of 8 M urea, and refolded and converted to an active molecule. The enzyme is refolded in a form with a putative prosequence
overproduction in Escherichia coli of an enzyme derivative with the mutation of the active-site serine residue to Cys (Pro-Tk-S359C), its derivatives lacking the N-propeptide (ProC-Tk-S359C) and both propeptides (Tk-S359C), and a His-tagged form of the C-propeptide (ProC*)
overproduction of pro-S255A in Escherichia coli BL21-Codon-Plus(DE3)
the propeptide and mature domain are independently overproduced in Escherichia coli
Tk-S359A without N- and C-propeptides is overproduced in Escherichia coli in a soluble form
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
to clarify the role of Ca2+ ions (Ca1, Ca6, and Ca7) Pro-TKS derivatives are constructed that lack the Ca1 ion (Pro-TKS/DELTACa1), Ca6 ion (Pro-TKS/DELTACa6), and Ca7 ion (Pro-TKS/DELTACa7), and their active site mutants (Pro-S324A/DELTACa1, Pro-S324A/DELTACa6, and Pro-S324A/DELTACa7, respectively). Pro-TKS/DELTACa6 and Pro-TKS/DELTACa7 fully mature into their active forms upon incubation at 80°C for 30 min as do Pro-TKS. The mature enzymes are as active as Tk-subtilisin at 80 °C, indicating that the Ca6 and Ca7 ions are not important for activity. Pro-TKS/DELTACa1 matures poorly at 80°C because of the instability of its mature domain. The enzymatic activity of Tk-subtilisin/DELTACa1 is determined to be 50% of that of Tk-subtilisin using the refolded protein. This result suggests that the Ca1 ion is required for the maximal activity of Tk-subtilisin. The refolding rates of all Pro-S324A derivatives are comparable to that of Pro-S324A (active site mutant of Pro-TKS), indicating that these Ca2+ ions are not needed for folding of Tk-subtilisin. The stabilities of Pro-S324A/DELTACa1 and Pro-S324A/DELTACa6 are decreased by 26.6 and 11.7°C, respectively, in Tm compared to that of Pro-S324A. The half-lives of Tk-subtilisin/DELTACa6 and Tk-subtilisin/DELTACa7 at 95°C are 8fold and 4fold lower than that of Tk-subtilisin, respectively. These results suggest that the Ca1, Ca6, and Ca7 ions, especially the Ca1 ion, contribute to the hyperthermostabilization of Tk-subtilisin. The counting of amino acids refers to the enzyme protein without the signal peptide (amino acid 1-24) and the propeptide (amino acid 25-106)
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
medicine
medicine
potential application for the inactivation of abnormal prion protein PrPSc, a protease-resistant isoform of normal prion protein. PrPSc is largely unaffected by standard methods of sterilization, thus, contaminated neurosurgical instruments are the major cause of human transmissible spongiform encephalopathies
medicine
the abnormal prion protein (scrapie-associated prion protein, PrP(Sc)) is considered to be included in the group of infectious agents of transmissible spongiform encephalopathies. Since PrP(Sc) is highly resistant to normal sterilization procedures, the decontamination of PrP(Sc) is a significant public health issue. The hyperthermostable protease, Tk-subtilisin, can be an effective agent for medical decontamination of of PrP(Sc). Rather small amounts of Tk-subtilisin (0.3 U) are required to degrade PrP(Sc) at 100 °C and pH 8.0. In addition, Tk-subtilisin is observed to degrade PrP(Sc) in the presence of sodium dodecyl sulfate or other industrial surfactants. Basic information for the practical use of the proteolytic enzyme for PrP(Sc) degradation is provided
medicine
the enzyme may have potential application as a detergent additive to decrease the infectivity of abnormal prion protein PrPSc
medicine
Tk-SP-containing detergents can be developed to decrease the secondary infection risks of transmissible spongiform encephalopathies
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Tanaka, S.; Saito, K.; Chon, H.; Matsumura, H.; Koga, Y.; Takano, K.; Kanaya, S.
Crystallization and preliminary X-ray diffraction study of an active-site mutant of pro-Tk-subtilisin from a hyperthermophilic archaeon
Acta Crystallogr. Sect. F
62
902-905
2006
Thermococcus kodakarensis (P58502)
Pulido, M.A.; Tanaka, S.; Sringiew, C.; You, D.J.; Matsumura, H.; Koga, Y.; Takano, K.; Kanaya, S.
Requirement of left-handed glycine residue for high stability of the Tk-subtilisin propeptide as revealed by mutational and crystallographic analyses
J. Mol. Biol.
374
1359-1373
2007
Thermococcus kodakarensis (P58502)
Takeuchi, Y.; Tanaka, S.; Matsumura, H.; Koga, Y.; Takano, K.; Kanaya, S.
Requirement of a unique Ca(2+)-binding loop for folding of Tk-subtilisin from a hyperthermophilic archaeon
Biochemistry
48
10637-10643
2009
Thermococcus kodakarensis (P58502)
Kannan, Y.; Koga, Y.; Inoue, Y.; Haruki, M.; Takagi, M.; Imanaka, T.; Morikawa, M.; Kanaya, S.
Active subtilisin-like protease from a hyperthermophilic archaeon in a form with a putative prosequence
Appl. Environ. Microbiol.
67
2445-2452
2001
Thermococcus kodakarensis (P58502)
Pulido, M.; Saito, K.; Tanaka, S.; Koga, Y.; Morikawa, M.; Takano, K.; Kanaya, S.
Ca2+-dependent maturation of subtilisin from a hyperthermophilic archaeon, Thermococcus kodakaraensis: the propeptide is a potent inhibitor of the mature domain but is not required for its folding
Appl. Environ. Microbiol.
72
4154-4162
2006
Thermococcus kodakarensis (P58502)
Koga, Y.; Tanaka, S.; Sakudo, A.; Tobiume, M.; Aranishi, M.; Hirata, A.; Takano, K.; Ikuta, K.; Kanaya, S.
Proteolysis of abnormal prion protein with a thermostable protease from Thermococcus kodakarensis KOD1
Appl. Microbiol. Biotechnol.
98
2113-2120
2013
Thermococcus kodakarensis (P58502)
Uehara, R.; Takeuchi, Y.; Tanaka, S.; Takano, K.; Koga, Y.; Kanaya, S.
Requirement of Ca(2+) ions for the hyperthermostability of Tk-subtilisin from Thermococcus kodakarensis
Biochemistry
51
5369-5378
2012
Thermococcus kodakarensis (P58502)
Tanaka, S.; Koga, Y.; Takano, K.; Kanaya, S.
Inhibition of chymotrypsin- and subtilisin-like serine proteases with Tk-serpin from hyperthermophilic archaeon Thermococcus kodakaraensis
Biochim. Biophys. Acta
1814
299-307
2010
Thermococcus kodakarensis (P58502)
Hirata, A.; Hori, Y.; Koga, Y.; Okada, J.; Sakudo, A.; Ikuta, K.; Kanaya, S.; Takano, K.
Enzymatic activity of a subtilisin homolog, Tk-SP, from Thermococcus kodakarensis in detergents and its ability to degrade the abnormal prion protein
BMC Biotechnol.
13
19
2013
Thermococcus kodakarensis (P58502), Thermococcus kodakarensis
Sinsereekul, N.; Foophow, T.; Yamanouchi, M.; Koga, Y.; Takano, K.; Kanaya, S.
An alternative mature form of subtilisin homologue, Tk-SP, from Thermococcus kodakaraensis identified in the presence of Ca2+
FEBS J.
278
1901-1911
2011
Thermococcus kodakarensis (P58502), Thermococcus kodakarensis
Uehara, R.; Ueda, Y.; You, D.J.; Koga, Y.; Kanaya, S.
Accelerated maturation of Tk-subtilisin by a Leu->Pro mutation at the C-terminus of the propeptide, which reduces the binding of the propeptide to Tk-subtilisin
FEBS J.
280
994-1006
2013
Thermococcus kodakarensis (P58502)
Tanaka, S.; Takeuchi, Y.; Matsumura, H.; Koga, Y.; Takano, K.; Kanaya, S.
Crystal structure of Tk-subtilisin folded without propeptide: requirement of propeptide for acceleration of folding
FEBS Lett.
582
3875-3878
2008
Thermococcus kodakarensis (P58502)
Foophow, T.; Tanaka, S.; Angkawidjaja, C.; Koga, Y.; Takano, K.; Kanaya, S.
Crystal structure of a subtilisin homologue, Tk-SP, from Thermococcus kodakaraensis: requirement of a C-terminal beta-jelly roll domain for hyperstability
J. Mol. Biol.
400
865-877
2010
Thermococcus kodakarensis (P58502), Thermococcus kodakarensis
Yuzaki, K.; Sanda, Y.; You, D.J.; Uehara, R.; Koga, Y.; Kanaya, S.
Increase in activation rate of Pro-Tk-subtilisin by a single nonpolar-to-polar amino acid substitution at the hydrophobic core of the propeptide domain
Protein Sci.
22
1711-1721
2013
Thermococcus kodakarensis (P58502)
Okada, J.; Koga, Y.; Takano, K.; Kanaya, S.
Slow unfolding pathway of hyperthermophilic Tk-RNase H2 examined by pulse proteolysis using the stable protease Tk-subtilisin
Biochemistry
51
9178-9191
2012
Thermococcus kodakarensis (P58502)
Uehara, R.; Angkawidjaja, C.; Koga, Y.; Kanaya, S.
Formation of the high-affinity calcium binding site in pro-subtilisin E with the insertion sequence IS1 of pro-Tk-subtilisin
Biochemistry
52
9080-9088
2013
Thermococcus kodakarensis (P58502)
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