酶架构:氨基酸侧链的功能是优化三酸异构酶活性位点的基性
Xiang Zhai1, Christopher J Reinhardt2, M Merced Malabanan3
1Department of Chemistry , University at Buffalo, SUNY , Buffalo , New York 14260-3000 United States.
Journal of the American Chemical Society
|June 5, 2018
概括
这项研究研究了三酸盐异构酶 (TIM) 动力学和糖酸三的抑制. 改变TIM的突变
科学领域:
- 生物化学
- 酶动力学
- 蛋白质工程
背景情况:
- 三酸盐异构酶 (TIM) 催化糖解的一个关键步骤.
- 了解TIM的催化机制和基质结合对于代谢途径研究至关重要.
- 像糖酸三这样的抑制剂提供了对酶活性部位相互作用的洞察力.
研究的目的:
- 描述野生型和突变型TIM的pH依赖动力学.
- 阐明特定氨基酸残留在TIM的催化活性和基质结合中的作用.
- 调查糖三的抑制机制.
主要方法:
- 酶运动测定测量 kcat 和 Km.
- 对糖酸三的抑制常数 (Ki) 的确定
- 对野生型和突变型TIM的pH值分析.
- 线性自由能量相关分析.
主要成果:
- 在糖三与TIM结合时,pH 概况显示了质子化事件.
- 在催化效率 (kcat/ Km) 和抑制剂结合性之间发现了强烈的相关性.
- 突变显著改变了主要活性部位残留物的催化活性和pKa.
- 特定的残留物 (E165,Y208,S211,I170,P166) 被认为对TIM的功能至关重要.
结论:
- 该研究支持一种特定氨基酸残留物稳定过渡状态并增强E165的基本性.
- 影响这些残留物的突变会影响催化效率和抑制剂结合.
- TIM的催化机制涉及精确定位活性部位残留物以促进基质异构.
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