过渡状态结构用于菌毒素催化NAD的水解
Paul J Berti1, Steven R Blanke, Vern L Schramm
1Contribution from the Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, and Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts 02115.
Journal of the American Chemical Society
|September 28, 2011
概括
使用动态同位素效应 (KIEs) 研究了NAD的二毒素 (DTA) 水解. 这揭示了协调的,类似氧碳离子的过渡状态,阐明了DTA的催化机制.
科学领域:
- 生物化学 生物化学
- 酶学 是一种酶学.
- 毒理学 毒理学 毒理学
背景情况:
- 双菌毒素 (DTA) 催化了使用NAD ((+)) 作为基质的真核延长因子2的ADP-ribosylation.
- 在缺乏其生理接受器的情况下,DTA将NAD ((+) 水解成ADP-ribose和尼古丁胺胺.
- 这种水解反应为通过动态同位素效应 (KIEs) 研究酶的催化机制提供了一条途径.
研究的目的:
- 确定由DTA催化的NAD (((+) 水解的过渡状态结构.
- 使用动态同位素效应 (KIE) 测量,阐明DTA催化NAD(+) 水解的机制.
- 了解DTA如何通过酶相互作用稳定过渡状态.
主要方法:
- 使用放射性标记方法测量同位素标记NAD分子的竞争性动态同位素效应 (KIEs).
- 使用全分子质谱法确定环氧 (4'-(18) O) KIE.
- 结构位和债券能量/债券边界振动分析,用于预测试验过渡状态结构的KIEs.
- 将确定过渡状态结构融入DTA的X射线晶体结构中.
主要成果:
- 测量了多个KIE用于各种标记NAD(+) 位置,观察到1'-(3) H (1.200 ± 0.005) 和2'-(3) H (1.142 ± 0.005) 的显著值.
- 通过放射标记和质谱测量测量的4'-(18) O KIE的平均值为0.988 ± 0.003.
- 实验KIE与对应的预测一致,高度类似氧碳离子的过渡状态,其残留键次序为0.02到离开组和0.03到核爱好者 (A(N) D(N) 机制).
- 将过渡状态插入DTA活性部位揭示了稳定机制,包括尼古丁胺环溶解和静电相互作用.
结论:
- DTA通过协同的A(N) D(N) 机制催化NAD(+) 水解,包括离开组和核友的参与.
- 过渡状态非常类似于氧化碳离子,通过DTA活性部位内的特定酶相互作用稳定.
- 这些发现为DTA介导的NAD () 解的催化机制和过渡状态稳定提供了详细的见解.
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