徳島大学 教育・研究者情報データベース(EDB)

1. 徳島大学.総合科学部.自然システム学科(1993年4月1日〜)

1. 佐藤 高則

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2. (英) Kohaku Yuko

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3. (英) Sugiyama Noriko

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4. (英) Yamaguchi Yasuaki

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5. (英) Kouzai Miho

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(英) Threonine 138 is crucial for Quaternary Structure and Thermostability of Thermus thermophilus Inorganic Pyrophosphatase.

(英) Inorganic pyrophosphatase (E.C. 3.6.1.1) from Thermus thermophilus (Tth PPase) forms the hexamer, and it was suggested from X-ray studies that its intersubunit interactions stabilize the whole molecule. However, the contribution of Thr138 at intertrimer interface to quaternary structure and thermostability was unknown functionally. Therefore, we prepared the Thr138-substituted variants (T138A, V, N, and H) by site-directed mutagenesis. Then, the enzyme activities of T138A and V were drastically decreased to 15 and 30 % of the wild type respectively. In particular, half of T138A hexamer were dissociated into trimers even in native state, and their thermostabilities of enzyme activity and quaternary structure were decreased relative to those of the wild type Tth PPase. On the other hand, T138N and T138H variants maintained the enzyme activity and hexamer in the native state, which thermostabilities of hexamer were higher than those of T138A and V. Moreover, reassociation of hexamer in T138H and N variants was possible by increasing the enzyme concentration up to 1 mg/ml. Therefore, we suggest that both methyl and polar groups in Thr138 of Tth PPase are crucial for thermostability and quaternary structure, and it may contribute to the formation of stable trimer-trimer interface.