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Literature summary extracted from

  • Foophow, T.; Tanaka, S.; Koga, Y.; Takano, K.; Kanaya, S.
    Subtilisin-like serine protease from hyperthermophilic archaeon Thermococcus kodakaraensis with N- and C-terminal propeptides (2010), Protein Eng. Des. Sel., 23, 347-355.
    View publication on PubMed

Activating Compound

EC Number Activating Compound Comment Organism Structure
3.4.21.62 additional information unlike subtilisin, subtilisin-like serine protease does not require propeptide for folding Thermococcus kodakarensis

Application

EC Number Application Comment Organism
3.4.21.62 biotechnology subtilisin-like serine protease has a great advantage over other proteases in high resistance to heat, denaturants, detergents and chelating agents and therefore has great potential for application in biotechnology fields Thermococcus kodakarensis

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.4.21.62 the gene encoding the 1689 derivative without a putative N-terminal signal sequence, Pro-subtilisin-like serine protease (Met + Ala1-Gly640), ligated into pET25b vector, mutant Pro-S359A overexpressed in Escherichia coli BL21-CodonPlus(DE3)-RIL cells Thermococcus kodakarensis

Protein Variants

EC Number Protein Variants Comment Organism
3.4.21.62 S359A active site mutant. Upon overproduction, Pro-S359A accumulates in the cells in a soluble form Thermococcus kodakarensis

Inhibitors

EC Number Inhibitors Comment Organism Structure
3.4.21.62 EDTA subtilisin exhibits little activity at 80°C in the presence of 10 mM. Subtilisin-like serine protease is fully active even in the presence of 10 mM EDTA Thermococcus kodakarensis
3.4.21.62 guanidine hydrochloride at 2 M inhibits subtilisin-like serine protease by 35% and at 4 M almost completely, whereas it has no inhibitory effect on subtilisin Thermococcus kodakarensis
3.4.21.62 additional information subtilisin-like serine protease is fully resistant to treatment with 2-5% SDS, 4-8 M urea, 10% Tween-20 or 10% Triton X-100. In gel assay, subtilisin-like serine protease is fully denatured prior to SDS-PAGE by trichloroacetic acid treatment, followed by boiling for 5 min in the presence of SDS Thermococcus kodakarensis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.4.21.62 0.11
-
 N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide subtilisin-like serine protease, at 20°C, in 50 mM Tris-HCl buffer, pH 7.5 Thermococcus kodakarensis
3.4.21.62 0.41
-
 N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide subtilisin-like serine protease, at 80°C, in 50 mM Tris-HCl buffer, pH 7.5 Thermococcus kodakarensis
3.4.21.62 2.4
-
 N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide subtilisin, at 20°C, in 50 mM Tris-HCl buffer, pH 8.0 Thermococcus kodakarensis
3.4.21.62 7.9
-
 N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide subtilisin, at 80°C, in 50 mM Tris-HCl buffer, pH 8.0 Thermococcus kodakarensis

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
3.4.21.62 additional information unlike subtilisin, subtilisin-like serine protease does not require Ca2+ for folding Thermococcus kodakarensis

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
3.4.21.62 44000
-
 Pro-subtilisin-like serine protease derivative with N- and/or C-terminal truncation, gel filtration Thermococcus kodakarensis
3.4.21.62 44200
-
 subtilisin-like serine protease, sequence analysis Thermococcus kodakarensis
3.4.21.62 55000
-
 Pro-subtilisin-like serine protease derivative with N- and/or C-terminal truncation, gel filtration Thermococcus kodakarensis
3.4.21.62 65000
-
 Pro-subtilisin-like serine protease, gel filtration Thermococcus kodakarensis
3.4.21.62 68630
-
 Pro-subtilisin-like serine protease, sequence analysis Thermococcus kodakarensis

Organism

EC Number Organism UniProt Comment Textmining
3.4.21.62 Thermococcus kodakarensis
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.4.21.62 subtilisin-like serine protease and Pro-S359A, at 4°C, by sonication, centrifugation, ammonium sulfate precipitation and gel filtration Thermococcus kodakarensis

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
3.4.21.62 510
-
 subtilisin-like serine protease Thermococcus kodakarensis
3.4.21.62 3100
-
 subtilisin Thermococcus kodakarensis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.4.21.62 azocasein + H2O
-
 Thermococcus kodakarensis ?
-
 ?
3.4.21.62 additional information autoprocessing of Pro-subtilisin-like serine protease Thermococcus kodakarensis ?
-
 ?
3.4.21.62 N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide + H2O
-
 Thermococcus kodakarensis N-succinyl-Ala-Ala-Pro-Phe + p-nitroaniline
-
 ?
3.4.21.62 oxidized insulin chain B + H2O is cleaved by subtilisin-like serine protease at multiple sites, preferably at the C-termini of the hydrophobic residues, such as Tyr, Phe, Leu, Val and Ala. This peptide is also cleaved by subtilisin at the C-termini of the hydrophobic residues, but at more specific sites Thermococcus kodakarensis ?
-
 ?

Subunits

EC Number Subunits Comment Organism
3.4.21.62 monomer 1* 42000, gel filtration, subtilisin-like serine protease. 1 * 65000, SDS-PAGE, Pro-S359A Thermococcus kodakarensis

Synonyms

EC Number Synonyms Comment Organism
3.4.21.62 subtilisin
-
 Thermococcus kodakarensis
3.4.21.62 subtilisin-like serine protease
-
 Thermococcus kodakarensis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.4.21.62 100
-
 subtilisin-like serine protease: 100°C, subtilisin: 90°C Thermococcus kodakarensis

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
3.4.21.62 80
-
 subtilisin-like serine protease is stable at 80°C for at least 3. It loses activity at 90°C and 100°C with half-lives of more than 3 h and 100 min, respectively. Subtilisin loses its activity at 100°C with a half-life of 50 min Thermococcus kodakarensis

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.4.21.62 1.6
-
 N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide subtilisin-like serine protease, at 20°C, in 50 mM Tris-HCl buffer, pH 7.5 Thermococcus kodakarensis
3.4.21.62 14
-
 N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide subtilisin, at 20°C, in 50 mM Tris-HCl buffer, pH 8.0 Thermococcus kodakarensis
3.4.21.62 25
-
 N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide subtilisin-like serine protease, at 80°C, in 50 mM Tris-HCl buffer, pH 7.5 Thermococcus kodakarensis
3.4.21.62 440
-
 N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide subtilisin, at 80°C, in 50 mM Tris-HCl buffer, pH 8.0 Thermococcus kodakarensis

pH Range

EC Number pH Minimum pH Maximum Comment Organism
3.4.21.62 7 11.5 subtilisin-like serine protease: pH 7.0-11.5, subtilisin: pH 8.0 and 11.5 Thermococcus kodakarensis

pH Stability

EC Number pH Stability pH Stability Maximum Comment Organism
3.4.21.62 2 12 subtilisin-like serine protease is fully stable between pH 7-11, whereas it is not fully stable below pH 6 and above pH 12. It loses more than 85% of its activity below pH 3 and at pH 13. Subtilisin is fully stable between pH 2-12 Thermococcus kodakarensis

General Information

EC Number General Information Comment Organism
3.4.21.62 physiological function Pro-subtilisin-like serine protease consists of an N-terminal propeptide (Ala1-Ala113), a mature domain (subtilisin-like serine protease, Val114-Val539) and a C-terminal propeptide (Asp540-Gly640) Thermococcus kodakarensis

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.4.21.62 6
-
 N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide subtilisin, at 20°C, in 50 mM Tris-HCl buffer, pH 8.0 Thermococcus kodakarensis
3.4.21.62 15
-
 N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide subtilisin-like serine protease, at 20°C, in 50 mM Tris-HCl buffer, pH 7.5 Thermococcus kodakarensis
3.4.21.62 56
-
 N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide subtilisin, at 80°C, in 50 mM Tris-HCl buffer, pH 8.0 Thermococcus kodakarensis
3.4.21.62 62
-
 N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide subtilisin-like serine protease, at 80°C, in 50 mM Tris-HCl buffer, pH 7.5 Thermococcus kodakarensis