Transient-State Kinetics of Apurinic/Apyrimidinic (AP) Endonuclease 1 Acting on an Authentic AP Site and Commonly Used Substrate Analogs: The Effect of Diverse Metal Ions and Base Mismatches
Transient-State Kinetics of Apurinic/Apyrimidinic (AP) Endonuclease 1 Acting on an Authentic AP Site and Commonly Used Substrate Analogs: The Effect of Diverse Metal Ions and Base Mismatches
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DOI：
10.1021/bi401218r
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发表时间：
2013-10-29
期刊：
影响因子：
2.9
通讯作者：
Delaney, Sarah
中科院分区：
文献类型：
--
作者：
Schermerhorn, Kelly M.;Delaney, Sarah
脱嘌呤/脱嘧啶核酸内切酶1（APE 1）是一种Mg 2+依赖性酶，负责切割DNA骨架5'端的脱嘌呤/脱嘧啶（AP）位点。在这里，我们使用快速淬火流（RQF）技术，以提供一个全面的动力学分析的链切割活性（k（化学））的APE 1作用于一个真实的AP网站沿着与两个广泛使用的类似物，减少AP网站和四氢呋喃（THF）网站。在存在生物相关Mg 2+的情况下，APE 1以比RQF的分辨率更快的速率切割所有三种底物，>= 700 s（-1）。为了获得k（化学）的定量值并便于真实底物与底物类似物的比较，我们用Mn 2+或Ni 2+代替Mg 2+或在损伤部位的5'端引入错配。这两种策略都足以减缓k（化学），并导致RQF分辨率内的速率。在获得定量速率的所有情况下，还原AP位点的k（化学）与真实AP位点无法区分。值得注意的是，相对于真实的AP位点，THF位点的k（化学）有小的降低，类似于1.5倍。这些结果突出了AP位点的C1'氧在链切割中的作用，并且在设计使用底物类似物的实验时值得考虑。
Apurinic/apyrimidinic endonuclease 1 (APE1) is an Mg2+-dependent enzyme responsible for incising the DNA backbone 5' to an apurinic/apyrimidinic (AP) site. Here, we use rapid quench flow (RQF) techniques to provide a comprehensive kinetic analysis of the strand-incision activity (k(chemistry)) of APE1 acting on an authentic AP site along with two widely used analogs, a reduced AP site and a tetrahydrofuran (THF) site. In the presence of biologically relevant Mg2+, APE1 incises all three substrates at a rate faster than the resolution of the RQF, >= 700 s(-1). To obtain quantitative values of k(chemistry) and to facilitate a comparison of the authentic substrate versus the substrate analogs, we replaced Mg2+ with Mn2+ or Ni2+ or introduced a mismatch 5' to the lesion site. Both strategies were sufficient to slow k(chemistry) and resulted in rates within the resolution of the RQF. In all cases where quantitative rates were obtained, k(chemistry) for the reduced AP site is indistinguishable from the authentic AP site. Notably, there is a small decrease, similar to 1.5-fold, in k(chemistry) for the THF site relative to the authentic AP site. These results highlight a role in strand incision for the C1' oxygen of the AP site and warrant consideration when designing experiments using substrate analogs.