在Hedgehog自动处理中保存活动并扩展基板宽容
Jing Zhao, Daniel A Ciulla1, Jian Xie
1Department of Chemistry , Binghamton University, State University of New York , Binghamton , New York 13902 , United States.
Journal of the American Chemical Society
|November 5, 2019
概括
在Hedgehog (Hh) 中的一种关键的阿斯巴酸突变自行处理为西丁 (D46H) 保持本源胆固醇活性并扩大基质耐受性. 这一发现为蛋白质工程和酶设计提供了洞察力.
科学领域:
- 生物化学
- 分子生物学
- 酶工程
背景情况:
- 刺 (Hh) 信号对于发育至关重要,并依赖于Hh前体蛋白自处理.
- 这种自动处理涉及胆固醇的修饰,由作为一般基的活性位点酸盐 (D46) 催化.
研究的目的:
- 研究D46在Hh自处理中的催化作用.
- 以其对胆固醇活性,基质特异性和活性部位构成的影响来描述D46H突变.
主要方法:
- 在体外研究中使用基于FRET的连续自动处理试验.
- 在细胞质验证中使用人类293T细胞的稳定表达.
- 应用NMR光谱来确定残留 pKa 值.
主要成果:
- D46H突变保留了与野生类型 (WT) 相似的原生胆固醇活性,具有相似的催化效率 (kmax/KM) 和pKa.
- D46H显著扩大了基质耐受性,对和的效率分别增加了200倍和300倍.
- 在D46H突变体中保留了活性部位的形状.
结论:
- D46H突变代表了Asp-to-His总基替代的第一个实例,既保留了蛋白质结构和总基活性.
- 在D46H中扩大基板耐受性是由于H46对四面体中间体的静电稳定.
- 这些发现为Hh自处理提供了基本的见解,并对蛋白质工程和酶设计产生了影响.
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