Information on EC 3.4.21.B57 - pernisine

Substrates: the prion protein PrP(Sc) is a pathological prion protein PrP isoform. The proteolytic activity of pernisine does not depend on the species of origin of the PrP used (bovine, mouse, human)
Products: -

abnormal prion protein PrP(Sc) + H2O

abnormal prion protein PrP(Sc) + H2O

Substrates: -
Products: -

abnormal prion protein PrP(Sc) + H2O

Substrates: prion protein PrP(Sc) is a pathological prion protein PrP isoform. The enzyme can disrupt PrPSc to a level undetectable by Western-blot analysis
Products: -

abnormal prion protein PrP(Sc) + H2O

Substrates: 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
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azocasein + H2O

Substrates: -
Products: -

azocasein + H2O

Substrates: -
Products: -

azocasein + H2O

Substrates: -
Products: -

azocasein + H2O

727248, 726708, 727468

Substrates: -
Products: -

azocasein + H2O

Substrates: -
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azocasein + H2O

Substrates: -
Products: -

Bovine serum albumin + H2O

Substrates: among the proteins tested, casein shows the highest degree of susceptibility with 100% of hydrolysis, while in the case of hemoglobin, ovalbumin, and bovine serum albumin it was about 50%
Products: -

Bovine serum albumin + H2O

casein + H2O

casein + H2O

Substrates: -
Products: -

casein + H2O

Hemoglobin + H2O

Hemoglobin + H2O

ovalbumin + H2O

ovalbumin + H2O

succinyl-Ala-Ala-Pro-Phe-4-nitroanilide + H2O

succinyl-Ala-Ala-Pro-Phe + 4-nitroaniline

Substrates: -
Products: -

succinyl-Ala-Ala-Pro-Phe-4-nitroanilide + H2O

succinyl-Ala-Ala-Pro-Phe + 4-nitroaniline

Substrates: -
Products: -

additional information

Substrates: pernisine has no activity on N-alpha-benzoyl-D-Arg-4-nitroanilide, N-alpha-benzoyl-D-Tyr-4-nitroanilide, and N-succinyl-Ala-Ala-Ala-4-nitroanilide used to detect trypsin, chymotrypsin, and elastase activity, respectively. Aminopeptidase activity is not detected
Products: -

additional information

Substrates: enzymatic degradation of protein aggregates by pernisine, such as for infective prions (PrPSc) from different origins (i.e., mouse, bovine, deer, human)
Products: -

additional information

Substrates: broad substrate specificity, with a slight preference for aromatic or large nonpolar P1 substrate residues
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additional information

Substrates: the propeptide is effectively degraded by the mature enzyme only at high temperatures, because it is too stable to be degraded at moderate temperatures
Products: -

additional information

Substrates: subtilisin shows a broad specificity but prefers to bind large non-beta-branched hydrophobic residues at the P1 position
Products: -

show all columns Go to Natural Substrate Search

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NATURAL SUBSTRATE

NATURAL PRODUCT

REACTION DIAGRAM

ORGANISM

UNIPROT

LITERATURE

COMMENTARY

REVERSIBILITY
r=reversible
ir=irreversible
?=not specified

pro-Tk-subtilisin + H2O

Tk-subtilisin + propeptide

Substrates: autoactivation
Products: -

proform pernisine + H2O

mature pernisine + signal sequence-N-terminal pro-region

Substrates: the enzyme performs autoproteolytical cleavage of its N-terminal pro-region for activation
Products: -

additional information

additional information

Substrates: enzymatic degradation of protein aggregates by pernisine, such as for infective prions (PrPSc) from different origins (i.e., mouse, bovine, deer, human)
Products: -

additional information

Substrates: the propeptide is effectively degraded by the mature enzyme only at high temperatures, because it is too stable to be degraded at moderate temperatures
Products: -

show all columns Go to Metals and Ions Search

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METALS and IONS

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

Ca2+

7 entries

CaCl2

NaCl

the maximal activity of the purified pernisine seen for 20 mM NaCl in the absence of 1 mM CaCl2 increases to more than 2fold after the addition of CaCl2, with even larger relative enhancement by CaCl2 at higher NaCl concentrations

additional information

NaCl and CaCl2 together do not show any cumulative effects, as they appear to separately affect the activity

Ca2+

1 mM, enhances activity. An increase in CaCl2 concentration did not affect further the enzyme activity

Ca2+

required for enzyme stability at higher temperatures, bound Ca2+ increases the thermostability of the subtilases or protects them from autolysis

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+

two Ca2+ ions bind to the beta-jelly roll domain

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+

bound at the high-affinity Ca1 site and low-affinity Ca2 site

731311

Ca2+

dependent on, subtilisin has high and low affinity calcium binding sites in its structure

CaCl2

maximum enhancement of the purified pernisine activity is observed at around 1 mM CaCl2. Further increasing the CaCl2 above 1 mM leads to a gradual decline in this enhanced activity. In the presence of 1 mM CaCl2, the proteolytic activity of pernisine is enhanced, to reach around 75% greater activity, with the maximum activity at 105.0°C

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

show all columns Go to Inhibitor Search

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INHIBITOR

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

IMAGE

2-mercaptoethanol

4-hydroxymercuribenzoate

10 mM, 80% inhibition

AMPHITOL 20Y-B

presence of 1% (w/v) AMPHITOL 20Y-B causes strong inhibitions, resulting in an enzyme retaining only 50% of its activity

Aprotinin

DFP

diisopropyl fluorophosphate

dithiothreitol

5 mM, 64% loss of activity in absence of CaCl2

DTT

42% and 52% inhibition at 1 mm and 5 mM

EDTA

EGTA

Guanidine HCl

5 mM, 65% loss of activity in absence of CaCl2, activation in presence of 1 mM CaCl2

iodoacetamide

slight inhibition

ovomucoid

phenylmethane sulfonylfluoride

PMSF

PMSF

SDS

Soybean trypsin inhibitor

1 mg/ml, complete inhibition

subtilisin propeptide

hyperthermophilic archaeon Thermococcus kodakaraensis has a subtilisin propeptide that inhibits the enzyme activity of mature subtilisin noncompetitively with a Ki value of 25 nM at 20°C

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

727071

Thermococcus kodakaraensis subtilisin propeptide

potent noncompetitive inhibitor of the mature domain

Urea

5 mM, 50% loss of activity in absence of CaCl2

additional information

2-mercaptoethanol

5 mM, 61% loss of activity in absence of CaCl2

2-mercaptoethanol

49% and 62% inhibition at 1 mm and 5 mM

Aprotinin

Aprotinin

EDTA

1 mM, 90% inhibition

EDTA

5 mM, complete loss of activity

EDTA

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; activity decreases with the increasing concentration of EDTA from 0.01 to 1% (w/v)

EGTA

1 mM, 94% inhibition

EGTA

5 mM, complete loss of activity

EGTA

PMSF

1 mM, 90% inhibition

PMSF

5 mM, complete loss of activity

PMSF

SDS

3%, 80% loss of activity in absence of CaCl2

SDS

9% and 90% inhibition at 0.1% and 3%

additional information

TPCK and TLCK, respectively chymotrypsin and trypsin-like inhibitors do not affect the activity. 1,10-phenanthroline has no effect

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)

show all columns Go to Activating Compound Search

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ACTIVATING COMPOUND

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

IMAGE

guanidinium hydrochloride

90% activation at 4 M

Urea

slight activation at 1-4 M

additional information

3 entries

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

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

show all columns Go to KM Value Search

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KM VALUE [mM]

SUBSTRATE

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

IMAGE

4 - 8

N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide

N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide

20°C, pH 9.5

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

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TURNOVER NUMBER [1/s]

SUBSTRATE

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

IMAGE

26 - 290

N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide

N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide

20°C, pH 9.5

290

N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide

80°C, pH 9.5

show all columns Go to Ki Value Search

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Ki VALUE [mM]

INHIBITOR

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

IMAGE

0.000025

subtilisin propeptide

pH not speficied in the publication, 20°C

0.000025

Thermococcus kodakaraensis subtilisin propeptide

20°C, pH 9.5

show all columns Go to Specific Activity Search

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SPECIFIC ACTIVITY [µmol/min/mg]

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

20°C, pH 9.5

140

80°C, pH 9.5

show all columns Go to pH Optimum Search

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pH OPTIMUM

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

6.5

in the presence of 1 mM CaCl2, at 92°C

6.8

native enzyme

7.5

8 - 9

9.5

in absence of CaCl2, at 92°C

recombinant codon-optimised wild-type enzyme

assay at

7.5

assay at

727248, 727468

7.5

assay at

9.5

9.5

assay at

show all columns Go to pH Range Search

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pH RANGE

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

3 - 10

active between pH 3.0 and pH 10.0, in the presence of 1 mM CaCl2, at 92°C

3.5 - 8

active between pH 3.5 and pH 8.0, in absence of CaCl2, at 92°C

4.5 - 9.1

the recombinant enzyme shows more than 90% activity within this pH range

6 - 12

pH 6.0: about 50% of maximal activity, pH 12.0: about 40% of maximal activity

show all columns Go to Temperature Optimum Search

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TEMPERATURE OPTIMUM

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

100

recombinant codon-optimised wild-type enzyme

105

assay at

substrate: azocasein, in absence of CaCl2

105

substrate: azocasein, in the presence of 1 mM CaCl2

105

native enzyme

assay at

727248, 727468

assay at

assay at

in the presence of 1 mM CaCl2

show all columns Go to Temperature Range Search

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TEMPERATURE RANGE

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

40 - 120

activity range

40 - 90

the enzyme exhibits 10% to 20% of the maximal activity at 40°C or 90°C

58 - 99

active between 58 °C and 99 °C

60 - 120

active in a broad range of temperature, 60°C: about 35% of maximal activity, 120°C: 80% of maximal activity

show all columns Go to Organism Search

only manually annotated data include textmining data from AMENDAenzyme occurrence in organism, localization and source tissue with reliability ranks include all textmining data from AMENDAenzyme occurrence in organism, localization and source tissue with reliability ranks + FRENDAco-occurrence of enzyme names and organism names (less precise)

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ORGANISM

COMMENTARY

LITERATURE

UNIPROT

SEQUENCE DB

SOURCE

727459, 728576

brenda

brenda

UniProt

brenda

729225, 731311, 731774, 769821

SwissProt

brenda

sequence including singnal peptide (amino acid 1-24) and propeptide (amino acid 25-106)

684164, 688387, 707480, 726706, 726708, 726790, 726988, 727071, 727248, 727468, 727484, 727510, 728144, 728762

SwissProt

brenda

show all columns Go to Localization Search

only manually annotated data include textmining data from AMENDAenzyme occurrence in organism, localization and source tissue with reliability ranks

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LOCALIZATION

ORGANISM

UNIPROT

COMMENTARY

GeneOntology No.

LITERATURE

SOURCE

brenda

brenda

brenda

extracellular

the enzyme is secreted into the external medium, probably as inactive pro-enzyme, with the assistance of the signal sequence

brenda

extracellular

brenda

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Highest Expressing Human Cell Lines

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show all columns Go to General Information Search

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GENERAL INFORMATION

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

evolution

subtilisin enzymes occur in bacteria, plants, and yeasts, and are Ser proteases that contain catalytic triad in order of aspartic acid, histidine, and Ser residues that have independently evolved and are different from the catalytic triad (histidine, aspartic acid, and Ser) order in the sequence of chymotrypsin and trypsin family

malfunction

when the aspartic residue at the active site of subtilisin is substituted with asparagine, there is an absence of processing by the mutated prosubtilisin via in vitro experiments, which suggests that propeptide acts as an intramolecular regulator for activation of subtilisin

physiological function

enzymatic degradation of protein aggregates by pernisine, such as for infective prions (PrPSc) from different origins (i.e., mouse, bovine, deer, human)

additional information

3 entries

additional information

pernisine is a subtilisin-like serine protease (i.e., a subtilase) with the catalytic triad of Asp149, His184 and Ser355. Catalytic nucleophile Ser355 has an important role in pernisine activity, but not in its activation process

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

additional information

subtilisin is composed of 268-275 amino acid residues and has a mass in the range of 26.9-38 kDa. It is generally secreted in 381 residues precursor form designated as preprosubtilisin, with the 29-residue signal peptide and the 275-residue mature subtilisin flanking the 77-residue propeptide in between. The propeptide domain is suggested as necessary for structural activity and the regulation of subtilisin. The domain is dubbed as the intramolecular chaperone, which helps the folding of protein for formation of an active 3D structure, though it is not part of the mature segment of subtilisin and does not add to subtilisin function

Go to AA Sequence and Transmembrane Helices SearchGo to Localization Prediction

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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).

P58502 pBLAST

TKSU_THEKO

Thermococcus kodakarensis (strain ATCC BAA-918 / JCM 12380 / KOD1)

422

43786

Swiss-Prot

Show Sequence

Secretory Pathway (Reliability: 1)

Q9YFI3 pBLAST

Q9YFI3_AERPE

Aeropyrum pernix (strain ATCC 700893 / DSM 11879 / JCM 9820 / NBRC 100138 / K1)

430

43714

TrEMBL

Show Sequence

other Location (Reliability: 2)

show all columns Go to Molecular Weight Search

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MOLECULAR WEIGHT

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

23000

x * 23000, SDS-PAGE, the purified pernisine is in active mature form at 23 kDa and in pre-form at 34 kDa, it appears that the purified pernisin is a mixture of two different active forms

34000

35160

gel filtration

36000

x * 36000, activated mature codon-optimised wild-type enzyme, SDS-PAGE, x * 55000, recombinant His-tagged proform codon-optimised wild-type enzyme, SDS-PAGE

40000

gel filtration

42000

gel filtration

43783

1 * 43783, calculation from sequence

44000

45000

active-site mutant S255A of pro-Tk-subtilisin

55000

x * 36000, activated mature codon-optimised wild-type enzyme, SDS-PAGE, x * 55000, recombinant His-tagged proform codon-optimised wild-type enzyme, SDS-PAGE

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)

34000

1 * 34000 SDS-PAGE

34000

x * 34000, SDS-PAGE, the purified pernisine is in active mature form at 23 kDa and in pre-form at 34 kDa, it appears that the purified pernisin is a mixture of two different active forms

44000

1 * 44000, SDS-PAGE

44000

1 * 44000, mutant enzyme S359A, SDS-PAGE

show all columns Go to Subunit Search

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SUBUNIT

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

6 entries

monomer

6 entries

additional information

three beta-sheets in the form of a highly twisted seven-stranded parallel beta-sheet with two antiparallel beta-sheets and nine alpha-helices make up the secondary structure of subtilisin

x * 23000, SDS-PAGE, the purified pernisine is in active mature form at 23 kDa and in pre-form at 34 kDa, it appears that the purified pernisin is a mixture of two different active forms

x * 34000, SDS-PAGE, the purified pernisine is in active mature form at 23 kDa and in pre-form at 34 kDa, it appears that the purified pernisin is a mixture of two different active forms

x * 36000, activated mature codon-optimised wild-type enzyme, SDS-PAGE, x * 55000, recombinant His-tagged proform codon-optimised wild-type enzyme, SDS-PAGE

x * 23000, SDS-PAGE, the purified pernisine is in active mature form at 23 kDa and in pre-form at 34 kDa, it appears that the purified pernisin is a mixture of two different active forms

x * 34000, SDS-PAGE, the purified pernisine is in active mature form at 23 kDa and in pre-form at 34 kDa, it appears that the purified pernisin is a mixture of two different active forms

x * 66000, Pro-Tk-S359C (an enzyme derivative with the mutation of the active-site serine residue to Cys)

monomer

1 * 34000 SDS-PAGE

monomer

1 * 34000 SDS-PAGE

monomer

1 * 43783, calculation from sequence

monomer

1 * 44000, SDS-PAGE

monomer

1 * 45000, active-site mutant S255A of pro-Tk-subtilisin, SDS-PAGE

monomer

1 * 44000, mutant enzyme S359A, SDS-PAGE

show all columns Go to Posttranslational Modification Search

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POSTTRANSLATIONAL MODIFICATION

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

proteolytic modification

14 entries

proteolytic modification

the purified pernisine has a proregion that is autocleaved during maturation

proteolytic modification

the enzyme needs to be heat-activated for 1 h at 90°C in activation buffer containing 10 mM HEPES, 1 mM CaCl2, pH 8.0, through autoproteolytical cleavage of its N-terminal pro-region from the 55 kDa inactive proform to the 36 kDa active form. The cleavage site of the proregion appears to be between Gln92 and Ala93

proteolytic modification

the purified pernisine has a proregion that is autocleaved during maturation

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

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

688387

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

the enzyme is autoprocessed from its precursor with N- and C-propeptides

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

the enzyme matures from the inactive precursor, Pro-Tk-subtilisin (Pro-TKS), upon autoprocessing and degradation of the propeptide (Tkpro)

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

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

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

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

731311

proteolytic modification

the enzyme activates autocatalytically cleaving its propeptide dependent on the presence of calcium ions. The subtilisin propeptide that inhibits the enzyme activity of mature subtilisin noncompetitively with a Ki value of 25 nM at 20°C. The propeptide may be required as intramolecular chaperone to suppress denaturation of the later mature subtilisin molecules from mature subtilisin molecules that are activated earlier

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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

show all columns Go to Protein Variants Search

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PROTEIN VARIANTS

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

S355A

site-directed mutagenesis, catalytically inactive active site mutant

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

S255A

active-site mutant enzyme

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

9 entries

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

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

688387

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

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

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

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

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

generation of IS1-deletion mutants of S324A and S324C enzyme variants

show all columns Go to Temperature Stability Search

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TEMPERATURE STABILITY

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

100

110

120

purified recombinant activated enzyme, 50 mMTris-HCl, pH 8.0 with 1 mM CaCl2, 4 h, completely stable

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

20 min, stable in presence of CaCls, about 60% loss of activity in absence of CaCl2

3 entries

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+

88.3

Tm value of mutant enzyme S359A, 10 mM CaCl2

4 entries

additional information

100

half life: 60 min, in absence of Ca2+

100

half life: 7 min, in the presence of 50 mM CaCl2

100

the enzyme loses half of its activity in 50 min

110

half life: 40 min, in absence of Ca2+

110

purified recombinant activated enzyme, 50 mMTris-HCl, pH 8.0 with 1 mM CaCl2, 4 h, loss of 30% activity

120

half life: 30 min, in absence of Ca2+

120

purified recombinant activated enzyme, 50 mMTris-HCl, pH 8.0 with 1 mM CaCl2, 4 h, loss of 50% activity

purified recombinant activated enzyme, 50 mMTris-HCl, pH 8.0 with 1 mM CaCl2, 4 h, loss of 20% activity

half life: more than 60 min, in the presence of 50 mM CaCl2

stable for at least 3 h

20 min, stable in presence of CaCl2, about 80% loss of activity in absence of CaCl2

4 h, no loss of activity, in absence of Ca2+

half life: 20 min, in the presence of 50 mM CaCl2

the enzyme loses half of its activity in 9 h

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

Go to General Stability Search

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GENERAL STABILITY

ORGANISM

UNIPROT

LITERATURE

1 mM CaCl2 stabilizes the enzyme for up to 4 h at 120°C

enzyme Tk-SP in its native form is highly stable in nonionic surfactants and EDTA

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

less stable in the presence of QUARTAMIN 60 W

stable in the presence of 0.1% (w/v) AMPHITOL 20Y-B

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 is stabilized in the presence of the Ca2+

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

show all columns Go to Organic Solvent Search

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ORGANIC SOLVENT

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

2-mercaptoethanol

dithiothreitol

guanidine-HCl

SDS

urea

2-mercaptoethanol

5%, reduces the activity by 80%

2-mercaptoethanol

5%, reduces the activity by 80%

dithiothreitol

5%, reduces the activity

dithiothreitol

5%, reduces the activity

guanidine-HCl

4 mM, 14% residual activity

guanidine-HCl

4 mM, 14% residual activity

guanidine-HCl

Tk-subtilisin retains its specific activity in 6 M guanidine HCl after 60 min incubation at room temperature

SDS

the enzyme retains 20% proteolytic activity

SDS

the enzyme retains 20% proteolytic activity

SDS

Tk-subtilisin shows no significant reduction of specific activity after 60 min incubation at room temperature with 5% SDS

urea

4 mM, 44% residual activity

urea

4 mM, 44% residual activity

urea

Tk-subtilisin retains its specific activity in 8 M urea after 60 min incubation at room temperature

Go to Purification (commentary) Search

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PURIFICATION (Commentary)

ORGANISM

UNIPROT

LITERATURE

4 entries

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

recombinant His-tagged codon-optimised wild-type and codon-optimised S355A active site mutant from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration

726706, 684164, 726708, 727468

Go to Cloned (commentary) Search

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CLONED (Commentary)

ORGANISM

UNIPROT

LITERATURE

expression in Escherichia coli

expression in Escherichia coli BL21(DE3)

expression in Escherichia coli of pernisine, lacking the leader sequence a fusion protein with glutathione-S-transferase

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)

recombinant overexpression of and His-tagged wild-type (pernisinewt), codon-optimised (pernisineco), and codon-optimised S355A active site mutant (pernisineS355Aco), with or without additional GST-tag or maltose-binding-protein-tag, in Escherichia coli strain BL21(DE3) requires codon preference optimisation and de-novo DNA synthesis. Undetectable expression level of unmodified wild-type enzyme, method evaluation

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 in Escherichia coli

expression in Escherichia coli

Go to Expression Search

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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)

show all columns Go to Application Search

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APPLICATION

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

industry

subtilisin shows general properties of thermostability, tolerance to neutral to high pH, broad specificity, and calcium-dependent stability leading to applicability of subtilisin in various industries including detergent, food processing and packaging, synthesis of inhibitory peptides, therapeutic, and waste management applications. The applicability is enlarged by bioengineering

medicine

4 entries

medicine

Tk-SP-containing detergents can be developed to decrease the secondary infection risks of transmissible spongiform encephalopathies

medicine

the enzyme may have potential application as a detergent additive to decrease the infectivity of abnormal prion protein PrPSc

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

top print hide References Search

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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

902-905

2006

Thermococcus kodakarensis (P58502)

16946475

brenda

688387

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)

17988685

brenda

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

10637-10643

2009

Thermococcus kodakarensis (P58502)

19813760

brenda

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.

2445-2452

2001

Thermococcus kodakarensis (P58502)

11375149

brenda

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.

4154-4162

2006

Thermococcus kodakarensis (P58502)

16751527

brenda

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.

2113-2120

2013

Thermococcus kodakarensis (P58502)

23880875

brenda

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

5369-5378

2012

Thermococcus kodakarensis (P58502)

22686281

brenda

727071

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)

21112419

brenda

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.

2013

Thermococcus kodakarensis (P58502), Thermococcus kodakarensis

23448268

brenda

Catara, G.; Ruggiero, G.; La Cara, F.; Digilio, F.A.; Capasso, A.; Rossi, M.

A novel extracellular subtilisin-like protease from the hyperthermophile Aeropyrum pernix K1: biochemical properties, cloning, and expression

Extremophiles

391-399

2003

Aeropyrum pernix, Aeropyrum pernix DSM 11879

12908102

brenda

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

21443525

brenda

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)

23237738

brenda

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)

18951896

brenda

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

20595040

brenda

Snajder, M.; Vilfan, T.; Cernilec, M.; Rupreht, R.; Popovic, M.; Juntes, P.; Serbec, V.C., Ulrih, N.P.

Enzymatic degradation of PrPSc by a protease secreted from Aeropyrum pernix K1

PLoS One

e39548

2012

Aeropyrum pernix, Aeropyrum pernix DSM 11879

22761822

brenda

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.

1711-1721

2013

Thermococcus kodakarensis (P58502)

24115021

brenda

729225

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

9178-9191

2012

Thermococcus kodakarensis (P58502)

23106363

brenda

731311

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

9080-9088

2013

Thermococcus kodakarensis (P58502)

24279884

brenda

Uehara, R.; Tanaka, S.; Takano, K.; Koga, Y.; Kanaya, S.

Requirement of insertion sequence IS1 for thermal adaptation of Pro-Tk-subtilisin from hyperthermophilic archaeon

Extremophiles

841-851

2012

Thermococcus kodakarensis (P58502)

22996828

brenda

Snajder, M.; Mihelic, M.; Turk, D.; Ulrih, N.P.

Codon optimisation is key for pernisine expression in Escherichia coli

PLoS ONE

e0123288

2015

Aeropyrum pernix (Q9YFI3)

25856104

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