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Literature summary for 3.4.21.B57 extracted from

  • 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+ (2011), FEBS J., 278, 1901-1911.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
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*) Thermococcus kodakarensis

Protein Variants

Protein Variants Comment Organism
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+ Thermococcus kodakarensis
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 Thermococcus kodakarensis
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 Thermococcus kodakarensis
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 Thermococcus kodakarensis
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 Thermococcus kodakarensis

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
63000
-
gel filtration, Pro-Tk-S359C (an enzyme derivative with the mutation of the active-site serine residue to Cys) Thermococcus kodakarensis
66000
-
x * 66000, Pro-Tk-S359C (an enzyme derivative with the mutation of the active-site serine residue to Cys) Thermococcus kodakarensis

Organism

Organism UniProt Comment Textmining
Thermococcus kodakarensis P58502 sequence including singnal peptide (amino acid 1-24) and propeptide (amino acid 25-106)
-

Posttranslational Modification

Posttranslational Modification Comment Organism
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 Thermococcus kodakarensis

Purification (Commentary)

Purification (Comment) Organism
-
Thermococcus kodakarensis

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
azocasein + H2O
-
Thermococcus kodakarensis ?
-
?

Subunits

Subunits Comment Organism
? x * 66000, Pro-Tk-S359C (an enzyme derivative with the mutation of the active-site serine residue to Cys) Thermococcus kodakarensis

Synonyms

Synonyms Comment Organism
Tk-SP
-
Thermococcus kodakarensis

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
80
-
assay at Thermococcus kodakarensis

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
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+ Thermococcus kodakarensis
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+ Thermococcus kodakarensis
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+ Thermococcus kodakarensis
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+ Thermococcus kodakarensis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
assay at Thermococcus kodakarensis