Literature summary extracted from

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 (2007), J. Mol. Biol., 374, 1359-1373.

Application

EC Number	Application	Comment	Organism
3.4.21.62	additional information	Gly56 is required in stabilizing the propeptide fold. Stabilization of this fold leads to strong binding of the propeptide to subtilisin, high resistance of the propeptide to proteolytic degradation, and slow maturation of Pro-subtilisin	Thermococcus kodakarensis

Cloned(Commentary)

EC Number	Cloned (Comment)	Organism
3.4.21.62	pET25b derivatives for overproduction of Pro-G56W, Pro-G56E, G56W-propeptide, and G56E-propeptide. G56W-propeptide overproduced in soluble form, while G56E-propeptide overproduced in insoluble form. S324A-subtilisin overproduced in inclusion bodies	Thermococcus kodakarensis

Crystallization (Commentary)

EC Number	Crystallization (Comment)	Organism
3.4.21.62	by the sitting drop vapor diffusion method. Crystal structures of propeptides in complex with S324A-subtilisin. Conformation of the propeptide is altered by the mutation, such that nonglycine residues at position 56 assume a right-handed conformation and hydrophobic interactions at the core region decrease	Thermococcus kodakarensis

Protein Variants

EC Number	Protein Variants	Comment	Organism
3.4.21.B57	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	Thermococcus kodakarensis
3.4.21.62	G56E	Pro-G56E gives a halo at temperatures equal or higher than 60 °C	Thermococcus kodakarensis
3.4.21.62	G56F	Pro-G56F gives a halo only at temperatures equal or higher than 70 °C	Thermococcus kodakarensis
3.4.21.62	G56S	Pro-G56S gives a halo from 40-80°C	Thermococcus kodakarensis
3.4.21.62	G56W	Pro-G56W gives a halo only at 80 °C like the wild-type	Thermococcus kodakarensis
3.4.21.62	S324A	active-site mutant of Pro-subtilisin	Thermococcus kodakarensis

Inhibitors

EC Number	Inhibitors	Comment	Organism	Structure
3.4.21.62	propeptide	inhibition in a concentration-dependent manner. Wild-type propeptide is more potent than G56W-, G56S- and G56E-propeptide	Thermococcus kodakarensis

Metals/Ions

EC Number	Metals/Ions	Comment	Organism	Structure
3.4.21.62	Ca2+	-	Thermococcus kodakarensis

Organism

EC Number	Organism	UniProt	Comment	Textmining
3.4.21.B57	Thermococcus kodakarensis	P58502	sequence including singnal peptide (amino acid 1-24) and propeptide (amino acid 25-106)	-
3.4.21.62	Thermococcus kodakarensis	-	-	-

Posttranslational Modification

EC Number	Posttranslational Modification	Comment	Organism
3.4.21.B57	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	Thermococcus kodakarensis

Purification (Commentary)

EC Number	Purification (Comment)	Organism
3.4.21.62	G56W-propeptide and G56E-propeptide, S324A-subtilisin purified in denatured form	Thermococcus kodakarensis

Substrates and Products (Substrate)

EC Number	Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
3.4.21.62	casein + H2O	-	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	-	?

Synonyms

EC Number	Synonyms	Comment	Organism
3.4.21.B57	Tk-subtilisin	-	Thermococcus kodakarensis

Temperature Range [°C]

EC Number	Temperature Minimum [°C]	Temperature Maximum [°C]	Comment	Organism
3.4.21.62	40	80	-	Thermococcus kodakarensis

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Release 2023.1 (January 2023)