原子从基质中通过在核酸减少酶中的活性位点基从基质中抽取的动力学
Lisa Olshansky1, Arturo A Pizano, Yifeng Wei
1Department of Chemistry and Chemical Biology, Harvard University , 12 Oxford Street, Cambridge, Massachusetts 02138, United States.
这项研究通过使用光化学方法揭示了核酸减少酶 (RNR) 催化中的速度限制步骤. 由光发起的激素传播揭示了3'-C-H键裂变是质子合电子转移 (PCET) 中最慢的步骤.
科学领域:
- 生物化学和酶学 生物化学和酶学
- 蛋白质基的化学结构 蛋白质基的化学结构
- 生物能量学和电子转移
背景情况:
- рибо核酸减少酶 (RNR) 是必要的酶,可催化核酸转化为脱氧核酸.
- 一类RNR利用复杂的质子合电子转移 (PCET) 途径,涉及超过35 Å的氧化还原活性氨基酸.
- 形态变化可能会掩盖RNR中PCET的内在动力学.
研究的目的:
- 调查大肠杆菌Ia类RNR的PCET途径中限制速率的步骤.
- 使用光化学方法将PCET动力学与较慢的形状变化脱.
- 用光谱观察和量化关键的基质中间体和转移事件.
主要方法:
- 在特定地点将[Re(I]光氧化剂纳入修改后的RNR ([Re]-β2) 中.
- 用2,3,5-trifluorotyrosine替换氨酸Y356以使激素产生成为可能.
- 暂时吸收光谱法用于监测不同条件下的铁基衰变动力学 (基质,蛋白质变体,同位素标记).
主要成果:
- 铁基的光化学生成允许观察PCET事件.
- 增强的基质衰变,表明前进的PCET,仅在野生类型的RNR和未标记的基质中观察到.
- 基质的3'-C-H键被C439基分裂被确定为前进PCET中的速度限制步骤.
结论:
- 3'-C-H键裂解步骤的速率常数下界为 (1.4 ± 0.4) × 10^4 s(-1).
- 光化学启动提供了一个强大的工具来研究酶中的快速,否则无法访问的激进动力学.
- 这项研究阐明了控制RNR催化过程的关键活性部位化学成分.
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