通过带正电荷的键捐赠体选择性稳定可里斯马酸突变酶过渡状态
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
概括
在Bacillus subtilis chorismate mutase中用素替换素显著降低了酶活性. 这凸显了过渡状态的静电稳定对于高效的催化作用的重要作用.
科学领域:
- 生物化学 生物化学
- 酶动力学 酶动力学
- 蛋白质工程是一种蛋白质工程.
背景情况:
- 来自 Bacillus subtilis 的 AroH chorismate 突变酶催化了芳香氨基酸生物合成途径中的关键反应.
- 活性部位残留物阿基宁90 (Arg90) 对于催化非常重要,它与基质合金酸盐形成键.
研究的目的:
- 调查静电相互作用在AroH合乐酶突变酶的催化机制中的作用.
- 了解Arg90正电荷对过渡状态稳定性的贡献.
主要方法:
- 化学半合成被用来用素 (Cit) 取代野生型Arg90,从而创造了Arg90Cit变体.
- 通过测量触媒速率常数 (kcat) 和迈凯利斯常数 (Km) 来分析酶动力学,无论是野生类型的酶还是变体的酶.
- 酶抑制剂的相互作用被评估使用一种受结构约束的抑制剂.
主要成果:
- 在Arg90Cit变体中,kcat下降了104倍以上,Km增加了2.7倍,这表明催化效率显著降低.
- 该变体对模仿过渡状态抑制剂的亲和力仅减少了约6倍,这表明该抑制剂不能完全复制过渡状态的电荷分布.
- 这些发现表明,对基质的反应性构成的简单补充性对于催化不够.
结论:
- 电静电稳定极化过渡状态的阴离子键供体对于AroH胆化酸盐突变酶的高催化效率至关重要.
- 在过渡状态下,Arg90的正电荷在C-O键裂解过程中,在稳定氧原子上发展的负电荷方面发挥着至关重要的作用.
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