Shinoda H, Hattori M, Shimizu A, Samejima T, Satoh T
Department of Chemistry, College of Science and Engineering, Aoyama Gakuin University, Chitosedai, Setagaya-ku, Tokyo, 157-8572, Japan.
J Biochem. 1999 Jan;125(1):58-63. doi: 10.1093/oxfordjournals.jbchem.a022268.
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.
为了确定嗜热脂肪芽孢杆菌（Bst.）三聚体无机焦磷酸酶（PPase）[EC 3.6.1.1]中Val75在其寡聚结构中的作用，我们使用定点诱变技术制备了Val75分别被Ala、Phe、Leu、Ile、Lys、Gln和Asp取代的变体。结果，缬氨酸被Ala、Phe、Leu和Ile等疏水残基取代的变体（V75A、F、L和I）表现出与野生型酶几乎相同水平的酶活性和热稳定性，而被Lys、Gln和Asp等亲水残基取代的变体（V75K、Q和D）的酶活性和热稳定性则大幅降低。随着温度升高，V75K和V75D从三聚体快速解离为单体，而V75F、V75L和V75I的解离速度比野生型慢。热处理对V75A或V75Q的解离没有特别影响，但这些变体即使在天然状态下也会轻微解离。因此，我们得出结论，Val75可能位于单体之间的界面，其与周围环境的疏水相互作用可能在该酶的热稳定性和寡聚亚基相互作用中起关键作用。
