著作: Sugiyama Noriko/Kohaku Yuko/Kouzai Miho/Yamaguchi Yasuaki/[佐藤 高則]/A drastic decrease in thermostability of Thermus thermophilus Inorganic Pyrophosphatase by substitution of Cysteine 168 to hydrophobic amino acids./[生化学]
(英) A drastic decrease in thermostability of Thermus thermophilus Inorganic Pyrophosphatase by substitution of Cysteine 168 to hydrophobic amino acids.
(英) Thermus thermophilus Inorganic pyrophosphatase (Tth PPase) is comprised of homohexamer, and exhibits high thermostability. However, it had not been elucidated that C-terminal region may contribute to its thermostability of Tth PPase. Therefore, we focused on sole cysteine (Cys168) in molecule, and examined the contribution of this Cys residue to the thermostability of Tth PPase by substitution to hydrophobic amino acids. Firstly, we prepared the three Cys168-substituted variants (C168 L, I, and F) by site-directed mutagenesis, followed by examined their enzyme activities and oligomeric structure. In native state, though wild type Tth PPase and all variants maintained the hexameric state, enzyme activity of only C168F was decreased to 58% of the wild type. On the other hand, the thermostabilities of enzyme activity and quaternary structure in all variants were decreased drastically relative to those of the wild type. In particular, C168I variant was dissociated into trimer and monomer after heating even at 65°C, which is appeared to be the most unstable of all variants. Therefore, it was suggested that the microenvironment around Cys168 in Tth PPase must be hydrophilic and less bulky for its thermostability and structural integrity, and C-terminal region of Tth PPase may play a key role in the stability of whole molecule.
|年月日||必須||2004年 10月 14日|