Powering DNA repair through substrate-electrostatic interactions
Powering DNA repair through substrate-electrostatic interactions
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DOI：
10.1021/bi027014x
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发表时间：
2003-02-25
期刊：
影响因子：
2.9
通讯作者：
Stivers, JT
中科院分区：
文献类型：
--
作者：
Jiang, YL;Ichikawa, Y;Stivers, JT
由DNA修复酶尿嘧啶DNA糖基化酶（UDG）催化的反应通过涉及带正电荷的氧杂卡宾离子糖和尿嘧啶阴离子离去基团的前所未有的逐步机制进行。在这里，我们使用一种新的方法来评估的催化贡献的静电相互作用之间的四个基本的磷酸二酯基团的DNA底物和阳离子过渡态。我们的策略是用不带电荷的（R）-或（S）-甲基膦酸酯键（MeP）取代这些磷酸基团中的每一个。然后，我们比较了这些甲基膦酸酯取代对催化的破坏作用与它们对阳离子1-氮脱氧核糖（1-aza-dR（+））oxacarbenium离子类似物与UDG-尿嘧啶阴离子二元复合物结合的破坏作用。一系列MeP取代底物的log k（cat）/K-m与1-aza-dR（+）抑制剂结合的log K-D的关系图给出了单位斜率的线性相关性，证实了过渡态的电子特征类似于1-aza-dR+的电子特征，并且DNA的阴离子骨架用于过渡态稳定。我们估计，所有结合的磷酸二酯与底物的相互作用有助于6-8千卡/摩尔降低活化屏障，与UDG的16千卡/摩尔催化能力相比，这种稳定性是显着的。然而，与选择性地稳定带电过渡态的酶的基团不同，这些磷酸二酯基团在基态也强烈相互作用。据我们所知，这些结果提供了第一个实验证据的静电稳定的带电的酶的过渡态和中间体使用的阴离子骨架的DNA。
The reaction catalyzed by the DNA repair enzyme uracil DNA glycosylase (UDG) proceeds through an unprecedented stepwise mechanism involving a positively charged oxacarbenium ion sugar and uracil anion leaving group. Here we use a novel approach to evaluate the catalytic contribution of electrostatic interactions between four essential phosphodiester groups of the DNA substrate and the cationic transition state. Our strategy was to substitute each of these phosphate groups with an uncharged (R)- or (S)-methylphosphonate linkage (MeP). We then compared the damaging effects of these methylphosphonate substitutions on catalysis with their damaging effects on binding of a cationic 1-azadeoxyribose (1-aza-dR(+)) oxacarbenium ion analogue to the UDG-uracil anion binary complex. A plot of log k(cat)/K-m for the series of MeP-substituted substrates against log K-D for binding of the 1-aza-dR(+) inhibitors gives a linear correlation of unit slope, confirming that the electronic features of the transition state resemble that of the 1-aza-dR+, and that the anionic backbone of DNA is used in transition state stabilization. We estimate that all of the combined phosphodiester interactions with the substrate contribute 6-8 kcal/mol toward lowering the activation barrier, a stabilization that is significant compared to the 16 kcal/mol catalytic power of UDG. However, unlike groups of the enzyme that selectively stabilize the charged transition state by an estimated 7 kcal/mol, these phosphodiester groups also interact strongly in the ground state. To our knowledge, these results provide the first experimental evidence for electrostatic stabilization of a charged enzymatic transition state and intermediate using the anionic backbone of DNA.