EC Number | Crystallization (Comment) | Organism |
---|---|---|
3.4.21.B57 | crystallization of the Pro-S324A/DELTACa6 mutant enzyme using the sitting-drop vapor-diffusion method at 4°C | Thermococcus kodakarensis |
EC Number | Protein Variants | Comment | Organism |
---|---|---|---|
3.4.21.62 | additional information | construction of pro-enzyme derivatives lacking 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. Mutants Pro-TKS/DELTACa6 and Pro-TKS/DELTACa7 fully mature into their active forms upon incubation at 80°C for 30 min like the wild-type enzyme. In contrast, mutant Pro-TKS/DELTACa1 matures poorly at 80°C because of the instability of its mature domain. Refolding rates of all Pro-S324A derivatives are comparable to that of Pro-S324A (active site mutant of wild-type pro-enzyme), indicating that these Ca(2+) ions are not needed for folding of Tk-subtilisin | Thermococcus kodakarensis |
3.4.21.62 | S324A | site-directed mutagenesis, active site mutant | Thermococcus kodakarensis |
EC Number | Metals/Ions | Comment | Organism | Structure |
---|---|---|---|---|
3.4.21.B57 | 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 | Thermococcus kodakarensis | |
3.4.21.62 | Ca2+ | the enzyme contains 7 Ca2+ ions, 4 of which are responsible for folding requirement of Ca2+ ions for the hyperthermostability of Tk-subtilisin, the Ca1, Ca6, and Ca7 ions, especially the Ca1 ion, contribute to the hyperthermostabilization of Tk-subtilisin | Thermococcus kodakarensis |
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 | Q5JIZ5 | - |
- |
EC Number | Posttranslational Modification | Comment | Organism |
---|---|---|---|
3.4.21.B57 | proteolytic modification | the enzyme matures from the inactive precursor, Pro-Tk-subtilisin (Pro-TKS), upon autoprocessing and degradation of the propeptide (Tkpro) | Thermococcus kodakarensis |
3.4.21.62 | proteolytic modification | the enzyme matures from the inactive precursor, Pro-Tk-subtilisin (Pro-TKS), upon autoprocessing and degradation of the propeptide (Tkpro) | Thermococcus kodakarensis |
EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
3.4.21.B57 | azocasein + H2O | - |
Thermococcus kodakarensis | ? | - |
? |
EC Number | Synonyms | Comment | Organism |
---|---|---|---|
3.4.21.B57 | Tk-subtilisin | - |
Thermococcus kodakarensis |
3.4.21.62 | subtilisin-like serine protease | - |
Thermococcus kodakarensis |
3.4.21.62 | Tk-subtilisin | - |
Thermococcus kodakarensis |
EC Number | Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|---|
3.4.21.62 | 80 | - |
assay at | Thermococcus kodakarensis |
EC Number | Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|---|
3.4.21.62 | additional information | - |
requirement of Ca2+ ions for the hyperthermostability of Tk-subtilisin.The Ca1, Ca6, and Ca7 ions, especially the Ca1 ion, contribute to the hyperthermostabilization of Tk-subtilisin | Thermococcus kodakarensis |
EC Number | Organism | Comment | Expression |
---|---|---|---|
3.4.21.B57 | Thermococcus kodakarensis | 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) | additional information |