陽性電荷の水素結合ドナーにより,コリスマートミュータゼ移行状態の選択的安定化
Alexander Kienhöfer1, Peter Kast, Donald Hilvert
1Laboratorium für Organische Chemie, Swiss Federal Institute of Technology, ETH Hönggerberg, CH-8093 Zürich, Switzerland.
Journal of the American Chemical Society
|March 13, 2003
まとめ
バシルス・サブティリス・コリスマート・ミュータゼにおけるアルギニンをシトルリンに置き換えることで,酵素の活性が著しく低下した. これは,効率的な触媒化のために移行状態の静電安定化が果たす重要な役割を強調しています.
科学分野:
- バイオケミストリー バイオケミストリー
- 酵素の動力学について
- タンパク質エンジニアリングは,
背景:
- バシルス・サブティリスのAroHコリスマートミュータゼは,芳香性アミノ酸生物合成経路における重要な反応を触媒化する.
- 活性部位の残留物であるアルギニン90 (Arg90) は,基板コリスマートと水素結合を形成し,触媒作用に不可欠である.
研究 の 目的:
- AroH chorismate mutaseの触媒メカニズムにおける静電相互作用の役割を調査する.
- 移行状態の安定化に対するArg90の正電荷の貢献を理解する.
主な方法:
- 化学的半合成を用いて,野生型Arg90をシトルリン (Cit) で置き換えて,Arg90Citの変種が生まれた.
- 酵素動態は,ワイルド型および変異型酵素の両方の触媒速度定数 (kcat) とミカエリス定数 (Km) を測定することによって分析されました.
- 酵素阻害剤の相互作用は,形状的に制約された阻害剤を使用して評価されました.
主要な成果:
- Arg90Citの変種は,kcatの104倍以上の減少とKmの2.7倍の増加を示し,触媒効率が著しく低下したことを示しました.
- 移行状態を模倣する阻害剤に対するこの変異体の親和度は約6倍しか減少しなかったため,阻害剤が移行状態の電荷分布を完全に複製していないことを示唆している.
- これらの発見は,基質の反応性構造に対する単純な補完性は,触媒作用には不十分であることを示しています.
結論:
- カチオンの水素結合ドナーによる偏極化された移行状態の静電安定化は,AroHコリスマート変異酵素の高触媒効率に不可欠である.
- Arg90の陽性電荷は,過渡状態のC-O結合の割れの間,酸素原子に発生する負の電荷を安定させる上で重要な役割を果たします.
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