Requirement of Ca2+ Ions for the Hyperthermostability of Tk-Subtilisin from Thermococcus kodakarensis

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• 作者：Ryo Uehara ; Yuki Takeuchi ; Shun-ichi Tanaka ; Kazufumi Takano ; Yuichi Koga ; Shigenori Kanaya
• 刊名：Biochemistry
• 出版年：2012
• 出版时间：July 3, 2012
• 年：2012
• 卷：51
• 期：26
• 页码：5369-5378
• 全文大小：438K
• 年卷期：v.51,no.26(July 3, 2012)
• ISSN：1520-4995

文摘

Tk-subtilisin, a hyperthermostable subtilisin-like serine protease from Thermococcus kodakarensis, matures from the inactive precursor, Pro-Tk-subtilisin (Pro-TKS), upon autoprocessing and degradation of the propeptide (Tkpro). It contains seven Ca²⁺ ions. Four of them (Ca2鈥揅a5) are responsible for folding of Tk-subtilisin. In this study, to clarify the role of the other three Ca²⁺ ions (Ca1, Ca6, and Ca7), we constructed Pro-TKS derivatives lacking the Ca1 ion (Pro-TKS/螖Ca1), Ca6 ion (Pro-TKS/螖Ca6), and Ca7 ion (Pro-TKS/螖Ca7), and their active site mutants (Pro-S324A/螖Ca1, Pro-S324A/螖Ca6, and Pro-S324A/螖Ca7, respectively). Pro-TKS/螖Ca6 and Pro-TKS/螖Ca7 fully matured into their active forms upon incubation at 80 掳C for 30 min as did Pro-TKS. The mature enzymes were as active as Tk-subtilisin at 80 掳C, indicating that the Ca6 and Ca7 ions are not important for activity. In contrast, Pro-TKS/螖Ca1 matured poorly at 80 掳C because of the instability of its mature domain. The enzymatic activity of Tk-subtilisin/螖Ca1 was 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 were 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/螖Ca1 and Pro-S324A/螖Ca6 were decreased by 26.6 and 11.7 掳C, respectively, in T_m compared to that of Pro-S324A. The half-lives of Tk-subtilisin/螖Ca6 and Tk-subtilisin/螖Ca7 at 95 掳C were 8- and 4-fold 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.