LpxCにおける残留イオン化は,67Zn NMRスペクトロスコピーによって直接観察された
Andrew S Lipton1, Robert W Heck, Marcy Hernick
1Biological Sciences Division, Pacific Northwest National Laboratory, 902 Battelle Boulevard, Richland, Washington 99352, USA.
Journal of the American Chemical Society
|September 3, 2008
まとめ
pHは,Aquifex aeolicus LpxCの構造に影響する. (67) Zn NMRで示されているように. His265の突然変異は,このpH依存性を除去し,His265がHis265であることを示しています.
科学分野:
- バイオケミストリー バイオケミストリー
- 構造生物学 構造生物学とは
- バイオフィジックス 生物物理学
背景:
- Aquifex aeolicus LpxCは,リポポリサッカリド生物合成に関与する酵素です.
- LpxCのpHに依存する振る舞いを理解することは,その触媒メカニズムを解明するために極めて重要です.
- 酵素の活性部位には,その機能に不可欠な亜鉛イオンが含まれています.
研究 の 目的:
- 固体 (67) Zn NMRの野生型 (WT) とH265A変異種 Aquifex aeolicus LpxC.の線形によるpH依存度を調査する.
- イオン化状態がpHで変化する活性部位残留物を特定する.
- NMRの発見を酵素活性と計算モデルと相関させる.
主な方法:
- 固体 (67) Zn核磁共鳴 (NMR) スペクトロスコーピー. 固体 (67) Zn核磁共鳴 (NMR) スペクトロスコーピー. 固体 (67) Zn核磁共鳴 (NMR) スペクトロスコーピー.
- 量子力学/分子力学 (QM/MM) モデリング.
- 酵素動力学 (pKa値によって示唆される).
主要な成果:
- WT LpxCは,pHに依存する (67) Zn NMRスペクトルを示し,異なる種が異なるpH値で現れ,2つのpKa値に対応しています.
- QM/MMモデリングは,Glu78とHis265のイオン化状態がpHによって変化することを示唆しています.
- H265A変異体はpH独立のスペクトルを示し,Glu78はプロトン化され,水は亜鉛イオンに結合している.
結論:
- WT LpxCのpH依存は,活性部位残留物のイオン化に起因する,おそらくGlu78とHis265.
- His265は,酵素のpHに依存する構造変化において重要な役割を果たします.
- 水は,WTおよび変異酵素の両方において,研究されたpH範囲全体で亜鉛イオンに調整され続けます.
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