通过甘-3-酸脱酶催化化转移对内在初级动态同位素的结构反应效应
Archie C Reyes1, Tina L Amyes1, John P Richard1
1Department of Chemistry, University at Buffalo, SUNY , Buffalo, New York 14260-3000, United States.
在糖-3-脱酶 (GPDH) 中研究了初级动同位素效应 (1°DKIE). 这些效应与酶活性无关,表明不同激活剂的过渡状态相似.
科学领域:
- 生物化学
- 酶动力学
- 化学运动学
背景情况:
- 甘3-酸脱酶 (GPDH) 对细胞能量代谢至关重要.
- 了解GPDH中化物转移的机制是阐明其催化功能的关键.
- 离子激活剂 (X2-) 调节GPDH活性,但它们在催化机制中的精确作用需要进一步研究.
研究的目的:
- 确定由GPDH催化由NADL转化为糖 (GA) 的初级动同位素效应 (1°DKIE).
- 研究不同的二激活剂 (X2-) 如何影响1°DKIE和酶反应.
- 阐明GPDH活性部位的基态和过渡状态结合相互作用之间的关系.
主要方法:
- 测量1°DKIEs (kcat/KGA和kcat) 使用标记的NADL在一系列的酶活性.
- 离子激活剂的系统变化 (FPO32-,HPO32-,SO42-,HOPO32-,S2O32-) 和一个未激活的状态.
- 对动态数据的分析,以推断化物转移和酶激活剂相互作用的机制细节.
主要成果:
- 观察到的1°DKIEs始终在2. 4~2. 9左右,并且在很大程度上独立于使用的二激活剂.
- 对于kcat/KGA和kcat均发现了类似的1°DKIEs,表明化物转移是限制速率或部分限制速率的.
- 催化效率的差异 (kcat) 与激活器的过渡状态结合相关,而不是基本状态结合.
结论:
- 在各种GPDH激活器复合物中,转移的内在1°DKIE是相似的.
- 由激活剂影响的过渡状态稳定显著影响GPDH催化率 (kcat).
- 观察到的1°DKIEs比通常在涉及量子力学道的酶催化反应中看到的更小.
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