   3.4.21.B57 | more |
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) |
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