Hydrophobic interactions of Val75 are critical for oligomeric thermostability of inorganic pyrophosphatase from Bacillus stearothermophilus.
To determine the role of Val75 in the oligomeric structure of trimeric inorganic pyrophosphatase (PPase) [EC 3.6.1.1] from Bacillus stearothermophilus (Bst.), we used site-directed mutagenesis to prepare variants in which Val75 was replaced by Ala, Phe, Leu, Ile, Lys, Gln, and Asp. As a result, the variants in which valine is replaced by hydrophobic residues such as Ala, Phe, Leu, and Ile (V75A, F, L, and I) show almost the same level of enzyme activity and thermostability as the wild type enzyme, whereas variants with hydrophilic residue replacements such as Lys, Gln, and Asp (V75K, Q, and D) showed gross reductions in enzyme activity and thermostability. The dissociation of V75K and V75D from trimer to monomers occurred rapidly as the temperature rose, while V75F, V75L, and V75I dissociated more slowly than the wild type. There was no particular effect of heat treatment on the dissociation of V75A or V75Q, but these variants were slightly dissociated even in the native state. Thus, we conclude that Val75 may locate at the interface between the monomers and its hydrophobic interactions with its surroundings may play a key role in the thermostability and oligomeric subunit interactions of the enzyme.
Shinoda
H
H
Department of Chemistry, College of Science and Engineering, Aoyama Gakuin University, Chitosedai, Setagaya-ku, Tokyo, 157-8572, Japan.
Hattori
M
M
Shimizu
A
A
Samejima
T
T
Satoh
T
T
D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006358:Hot Temperature; D043564:Inorganic Pyrophosphatase; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011755:Pyrophosphatases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013696:Temperature; D014633:Valine
D011994:Recombinant Proteins; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D014633:Valine