Threonine 138 is crucial for the Quaternary Structure and the Thermostability of Thermus thermophilus Inorganic Pyrophosphatase.

Inorganic pyrophosphatase (EC. 3.6.1.1) from Thermus thermophilus (Tth PPase) forms the thermostable hexamer，and it was suggested from X-ray studies that its intersubunit interactions stabilize the whole molecule. However，the contribution of Thr138 at the intertrimer interface to quatemary structure and thermostability was unknown functionally. Therefore，we prepared four Thr138-substituted variants (T138A，V ，N ，and H) by site-directed mutagenesis. Then，thermostabilities of the enzyme activity and the quatemary structure for T138V and A were decreased relative to those of the wild type Tth PPase，whereas T138H and N variants remained much hexamer contents and the enzyme activity than T138V and A. Therefore，we suggest that the polar group in Thr138 of Tth PPase is more crucial than the methyl group for thermostability and quatemary structure，and it may contribute to the formation of stable trimer-trimer interface

Thermostabilization by the Improvement of Intertrimeric Residues in Thermus thermophilus Inorganic Pyrophosphatase.

Inorganic pyrophosphatase (EC. 3.6.1.1) from Thermus thermophilus (Tth PPase)is a thermostable homohexamer of 174 amino acids，and its intertrimer interface is formed mainly by the symmetric α-helix A between subunits. Amino acids and their interactions composing intertrimer interface are different in hexameric Family I PPases，and then it was deduced that Tth PPase showed high thermostability because of stabilizing this interface by interactions of these residues. In this study，we focused on Thr138 and Ala141 residues in intertrimer interface of Tth PPase to confirm the relationship between intertrimeric residues and thermostability， and then improved their combination to His and Asp/Glu (HD or HE variant). As results，the HD variant showed the highest thermostability of enzyme activity，fluorescence spectra, and quaternary structure in the wild type Tth PPase and all variants. Especially，about 38% of hexamer and almost 40% of enzyme activity were observed in HD variant after heating even at 85℃. Therefore，we suggested that the conversion to a set of ionic His138 and Asp141 at intertrimer interface had increased the thermostability of Tth PPase，and then suppressed its thermal aggregation

Effect of Cys168 substitutions on the Thermostability and the Thermal Aggregation of Thermus thermophilus Inorganic Pyrophosphatase.

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