Literature summary for 3.4.21.62 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.

Search for more literature for 3.4.21.62

Activating Compound

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

Application

Application	Comment	Organism
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)

Cloned (Comment)	Organism
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

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

Inhibitors

Inhibitors	Comment	Organism	Structure
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
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
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]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
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
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
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
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

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

Molecular Weight [Da]

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

Organism

Organism	UniProt	Comment	Textmining
Thermococcus kodakarensis	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
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]

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

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
azocasein + H2O	-	Thermococcus kodakarensis	?	-	?
additional information	autoprocessing of Pro-subtilisin-like serine protease	Thermococcus kodakarensis	?	-	?
N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide + H2O	-	Thermococcus kodakarensis	N-succinyl-Ala-Ala-Pro-Phe + p-nitroaniline	-	?
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

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

Synonyms

Synonyms	Comment	Organism
subtilisin	-	Thermococcus kodakarensis
subtilisin-like serine protease	-	Thermococcus kodakarensis

Temperature Optimum [°C]

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

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
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]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism	Structure
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
14	-	N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide	subtilisin, at 20°C, in 50 mM Tris-HCl buffer, pH 8.0	Thermococcus kodakarensis
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
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

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

pH Stability

pH Stability	pH Stability Maximum	Comment	Organism
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

General Information	Comment	Organism
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]

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

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Release 2023.1 (January 2023)