通过67Zn NMR光谱直接观察到LpxC中的残留电离
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
概括
pH 影响了 Aquifex aeolicus LpxC 的结构,如 (67) Zn NMR 所示. 在His265中发生的一种突变消除了这种pH依赖,表明His265的pH依赖.
科学领域:
- 生物化学 生物化学
- 结构生物学 结构生物学
- 生物物理学的生物物理.
背景情况:
- 艾基菲克斯 (Aquifex aeolicus LpxC) 是一种参与脂多糖生物合成的酶.
- 了解LpxC的pH依赖性行为对于阐明其催化机制至关重要.
- 酶的活性部位含有离子,对其功能至关重要.
研究的目的:
- 为了研究固态 (67) Zn NMR野生型 (WT) 和H265A突变型Aquifex aeolicus LpxC.的线形的pH依赖性.
- 为了识别活性位点残留物,其离子化状态随着pH值变化而变化.
- 为了将NMR发现与酶活性和计算模型相关联.
主要方法:
- 固态 (67) Zn核磁共振 (NMR) 光谱学. 固态 (67) Zn核磁共振 (NMR) 光谱学. 固态 (67) Zn核磁共振 (NMR) 光谱学.
- 量子力学/分子力学 (QM/MM) 建模.
- 酶动力学 (暗示通过pKa值).
主要成果:
- WT LpxC表现出pH依赖的 (67) Zn NMR光谱,不同的物种出现在不同的pH值,对应于两个pKa值.
- QM/MM 建模表明,Glu78 和 His265 的电离状态随着 pH 的变化而变化.
- 突变H265A显示了pH独立的光谱,Glu78保持质子化,水与离子结合.
结论:
- WT LpxC的pH依赖性归因于活性部位残留物的电离,可能是Glu78和His265.
- His265在酶的pH依赖性结构变化中起着重要作用.
- 在WT和突变酶中,水在研究的pH范围内仍然与离子协调.
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