2つの活性部位残基の変異により,フォスファディチルイノシトール固有のフォスフォリファゼCをグルコースフォスファタゼに変換する
Jianwen Feng1, Kimberly Stieglitz, Mary F Roberts
1Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467, USA.
Journal of the American Chemical Society
|January 30, 2004
まとめ
2つのバクテリア酵素の変異により,新しいフォスファターゼが生まれました. この酵素は,元のフォスファディチルニノシトール特異のフォスフォリファーゼC型とは異なり,グルコース-6-フォスファートを効率的に分解します.
科学分野:
- バイオケミストリー バイオケミストリー
- 酵素学 酵素学とは
- 分子生物学は分子生物学である.
背景:
- バクテリアのフォスファティディル・イノシトール固有のフォスフォリファーゼC (PI-PLC) 酵素は通常,フォスフォニノシチドを水分解する.
- グルコース-6-ホスファタゼ酵素は,それぞれ異なるもので,特定の活性部位の要件を持っています.
研究 の 目的:
- 細菌PI-PLC変異体の酵素活性と基板特異性を調査する.
- 標的型変異によって酵素機能を変化させる可能性を調査する.
主な方法:
- サイト指向型変異は,細菌PI-PLC.にR69DおよびK115E変異を導入するために使用されました.
- 酵素活性アッセイは,イノシトール-1-フォスファート (I-1-P) とグルコース-6-フォスファート (G-6-P) を基質として使用して実施した.
- 観察された変化を合理化するために,バイオ情報分析とエネルギー最小化が採用されました.
主要な成果:
- ダブルミュータントのR69D/K115Eは,I-1-Pと比較して,G-6-Pに対するホスファターゼ活性が著しく高かった.
- 単一の変異R69DとK115Eは,有意なフォスファターゼ活性を与えない.
- バクテリアのPI-PLCは,既知のグルコース-6-フォスファタゼとのシーケンスホモロジーを示さなかった.
結論:
- 特定の変異は,細菌のPI-PLCを,グルコース-6-ホスファタゼ活性を持つ酵素に変換することができます.
- 変異した基板特異性は,酵素の触媒機構と活性部位の有意な変化を示唆しています.
- 合理的な酵素工学は,望ましい基板特異性を持つ新しい生物触媒を作り出すことができます.
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