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Measuring In Vitro ATPase Activity for Enzymatic Characterization
Published on: August 23, 2016
性酸酶催化是超敏感的电荷被扣留在活性位点离子之间的电荷
Ivana Nikolic-Hughes1, Patrick J O'brien, Daniel Herschlag
1Department of Chemical Engineering, Stanford University, Stanford, California 94305, USA.
Journal of the American Chemical Society
|June 30, 2005
概括
大肠杆菌性酸酶 (AP) 使用双金属活性位点来催化反应. 它的催化效率与基板电荷有很强的相关性,这表明它是一个高度敏感的静电环境.
科学领域:
- 生物化学 生物化学
- 酶动力学 酶动力学
- 蛋白质的静电学 蛋白质的静电学
背景情况:
- 埃舍里希亚大肠杆菌性酸酶 (AP) 是一种对酸单水解至关重要的双甲酶.
- 它的活性部位含有两个Zn2+离子,对于催化是必不可少的.
- 过渡状态涉及基质的氧原子和Zn2+金属集群之间的相互作用.
研究的目的:
- 为了研究基质的氧原子和AP双金属集群之间的相互作用的能量重要性.
- 量化基底电荷和AP催化能力之间的关系.
主要方法:
- 在基质的非桥接氧原子上的电荷的系统变化.
- 测定AP的催化活性在一系列具有不同电荷的基板上.
- 与氧原子电荷相关联的催化能力.
主要成果:
- 在AP催化能力和非桥梁氧原子上的电荷之间观察到强烈的线性相关性 (R2 = 0.98),跨越8个数量级.
- 催化能力显示出异常严重的依赖 (31 ± 2 kcal/mol/单元充电) 在这个充电.
- 这表明对带有差异电荷的基板存在重大歧视.
结论:
- AP的活性部位可能利用与双金属集群的静电相互作用来实现高的催化能力.
- 酶的活性部位具有高的静电潜力,使其对溶解基质的电荷高度敏感.
- 这种灵敏度为探测酶活性部位环境和理解酶演变提供了一种强大的方法.
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