ID 62132
Author
Kohaku, Yuko University of Tokushima
Maeda, Ayami University of Tokushima
Kouzai, Miho University of Tokushima
Sugiyama, Noriko University of Tokushima
Fukuhara, Ken-ichi Gakushuin University
Kanai, Taku University of Tokushima
Takei, Toshiaki Gakushuin University|The University of Tokyo
Miura, Kin-ichiro Gakushuin University|Chiba Institute of Technology|The University of Tokyo
Keywords
Inorganic pyrophosphatase
Thermal aggregation
Bulkiness Thermostability
Thermus thermophilus
Content Type
Departmental Bulletin Paper
Description
Thermus thermophilus Inorganic pyrophosphatase (Tth PPase) is comprised of homohexamer,and exhibits
high thermostability. However,the thermal aggregation containing the cross-linked dimer was observed after
heating above 85℃. Therefore,we focused on the sole cysteine (Cys168) in C-terminalregion,and evaluated the
effects of substitutions at this position on thermostability and thermal aggregation of Tth PPase.
Firstly,we prepared the four Cys168-substituted variants (C168A,L ,1,and F) by site-directed mutagenesis.
Although all variants formed hexamer in native state,C168A variant exhibited the highest thermostabilities for
the enzyme activity and quatemary structure in wild type and all variants,while the other variants decreased
them drastically as the side chain at the 168 position was much more bulky and hydrophobic in Tth PPase.
Moreover, suppression of thermal aggregation for C168A variant was observed in the ANS fluorescence
experiments. Therefore,we suggest that the small volume and less hydrophobicity of side chain at 168 position
may contribute to the conformational thermostability, and substitution with Ala is the most suitable for
thermostabilization and suppression ofthermal aggregation of Tth PPase.
Journal Title
徳島大学総合科学部自然科学研究 = Natural Science Research, The University of Tokushima
ISSN
09146385
NCID
AN10065859
Publisher
徳島大学総合科学部
Volume
22
Start Page
75
End Page
84
Sort Key
75
Published Date
2008-12-25
EDB ID
FullText File
language
eng
departments
Science and Technology