一个依赖PLP的种族酶通过单个活性位子突变转化为阿尔多酶
Florian P Seebeck1, Donald Hilvert
1Laboratorium für Organische Chemie, Swiss Federal Institute of Technology, ETH Hönggerberg, CH-8093 Zürich, Switzerland.
Journal of the American Chemical Society
|August 21, 2003
概括
氨酸赛马酶 (Alr) 突变将其转化为一种高效的阿尔多酶. Y265A突变酶选择性地分裂β-氨酸,提供了对酶进化和细菌细胞壁合成的见解.
科学领域:
- 生物化学 生物化学
- 酶学 是一种酶学.
- 结构生物学 结构生物学
背景情况:
- 氨酸种族酶 (Alr) 是一种依赖于酸盐的酶,对细菌细胞壁生物合成至关重要.
- Alr催化了氨酸的立体化学逆转,这是糖素形成的重要一步.
研究的目的:
- 为了研究在Geobacillus stearothermophilus alanine racemase中发生突变的铁素265 (Tyr265) 的功能后果.
- 描述Y265A突变的酶活性和基质特异性,探索其潜在的阿尔多酶活性.
主要方法:
- 使用局部导向的突变发生法来产生氨酸种族酶的Y265A突变体.
- 进行了酶动力学和立体选择性测试,以分析野生类型和突变酶的催化效率和基质偏好.
- 进行了野生类型和突变酶活动的比较分析.
主要成果:
- Y265A突变显著提高了酶的催化效率为β-氨酸裂2.3 x 10 ^ 5倍,将Alr转化为有效的阿尔多酶.
- 在Y265A突变体中,种族酶活性显著下降.
- 突变酶对 (2R,3S) - 氨酸具有很高的立体选择性,只处理D-氨基酸反体.
结论:
- Y265A突变表明,氨酸赛马酶可以很容易地转化为立体选择性阿尔多酶.
- 拟议的histidine 166 (His166) 在突变的逆醇反应中起到催化基的作用.
- 这些发现支持氨酸种族酶和D-氨酸阿尔多酶的共同进化起源.
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