Centre for Chemical Biology, Department of Chemistry, University of Sheffield, Sheffield S3 7HF, UK.
Biochemistry. 2010 Sep 21;49(37):8085-93. doi: 10.1021/bi100895j.
During replication and repair flap endonucleases (FENs) catalyze endonucleolytic and exonucleolytic (EXO) DNA hydrolyses. Altering the leaving group pK(a), by replacing the departing nucleoside with analogues, had minimal effect on k(cat)/K(M) in a T5FEN-catalyzed EXO reaction, producing a very low Brønsted coefficient, β(lg). Investigation of the viscosity dependence of k(cat)/K(M) revealed that reactions of EXO substrates are rate limited by diffusional encounter of enzyme and substrate, explaining the small β(lg). However, the maximal single turnover rate of the FEN EXO reaction also yields a near zero β(lg). A low β(lg) was also observed when evaluating k(cat)/K(M) for D201I/D204S FEN-catalyzed reactions, even though these reactions were not affected by added viscogen. But an active site K83A mutant produced a β(lg) = -1.2 ± 0.10, closer to the value observed for solution hydrolysis of phosphate diesters. The pH-maximal rate profiles of the WT and K83A FEN reactions both reach a maximum at high pH and do not support an explanation of the data that involves catalysis of leaving group departure by Lys 83 functioning as a general acid. Instead, a rate-limiting physical step, such as substrate unpairing or helical arch ordering, that occurs after substrate association must kinetically hide an inherent large β(lg). It is suggested that K83 acts as an electrostatic catalyst that stabilizes the transition state for phosphate diester hydrolysis. When K83 is removed from the active site, chemistry becomes rate limiting and the leaving group sensitivity of the FEN-catalyzed reaction is revealed.
在复制和修复过程中，核酸内切酶（FENs）催化核酸内切和核酸外切（EXO）的 DNA 水解反应。通过用类似物替换离去基团，改变离去核苷酸碱基的 pK(a)，对 T5FEN 催化的 EXO 反应中 k(cat)/K(M)的影响很小，产生非常低的 Brønsted 系数β(lg)。对 k(cat)/K(M)的粘滞依赖性的研究表明，EXO 底物的反应受酶和底物扩散相遇的速率限制，这解释了较小的β(lg)。然而，FEN EXO 反应的最大单轮周转率也产生了接近零的β(lg)。即使加入粘性物也不会影响 D201I/D204S FEN 催化反应的 k(cat)/K(M)，也观察到低β(lg)。但在活性位点 K83A 突变体中产生的β(lg) = -1.2 ± 0.10，更接近观察到的磷酸二酯盐溶液水解的β(lg)值。WT 和 K83A FEN 反应的 pH 最大速率曲线在高 pH 下均达到最大值，并且不支持涉及 Lys 83 作为广义酸催化离去基团离去的解释数据。相反，动力学上必须隐藏一个固有的大β(lg)，例如在底物结合后发生的底物解配对或螺旋拱排序的限速物理步骤。建议 K83 作为静电催化剂，稳定磷酸二酯水解的过渡态。当 K83 从活性位点中去除时，化学成为限速步骤，并且揭示了 FEN 催化反应的离去基团敏感性。
