Kinetic and mechanistic analysis of nonenzymatic, template-directed oligoribonucleotide ligation
Kinetic and mechanistic analysis of nonenzymatic, template-directed oligoribonucleotide ligation
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DOI：
10.1021/ja953712b
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发表时间：
1996-04-10
影响因子：
15
通讯作者：
Szostak, JW
中科院分区：
文献类型：
--
作者：
Rohatgi, R;Bartel, DP;Szostak, JW
二价阳离子在焦磷酸盐激活的模板导向的寡核糖核苷酸连接机制中的作用已经被研究。反应速率对Mg 2+浓度的依赖性表明了一种动力学方案，其中Mg 2+离子必须在连接之前结合才能进行。Mn ~（2+）、Ca ~（2+）、Sr ~（2+）和Ba ~（2+）也能催化该反应。虽然Pb 2+和Zn 2+在没有其他二价离子的情况下不催化反应，但当在Mg 2+存在下加入时，它们显著调节反应速率，其中Pb 2+刺激反应（高达65倍）而Zn 2+抑制反应。连接速率的对数作为pH的函数线性增加，斜率为0.95，表明该反应涉及一个关键的去质子化步骤。在不同的二价金属离子催化剂（Mn ~（2+）> Mg ~（2+）> Ca ~（2+）> Sr ~（2+）= Ba ~（2+））下观察到的连接速率与其结合水分子的pK（a）值成反比。pH值曲线和这些相对连接速率表明了一种机制，其中位于连接点附近的金属结合的氢氧根离子促进催化，最有可能是通过羟基亲核试剂的去质子化。改变离去基团或攻击羟基的影响，以及寡核苷酸连接的大Δ S-双匕首值（约-20 eu），与缔合过渡态一致。
The role of divalent cations in the mechanism of pyrophosphate-activated, template-directed oligoribonucleotide ligation has been investigated. The dependence of the reaction rate on Mg2+ concentration suggests a kinetic scheme in which a Mg2+ ion must bind before ligation can proceed. Mn2+, Ca2+, Sr2+, and Ba2+ can also catalyze the reaction. Although Pb2+ and Zn2+ do not catalyze the reaction in the absence of other divalent ions, they significantly modulate the reaction rate when added in the presence of Mg2+, With Pb2+ stimulating the reaction (up to 65-fold) and Zn2+ inhibiting the reaction. The logarithm of the ligation rate increases linearly, with slope of 0.95, as a function of pH, indicating that the reaction involves a single critical deprotonation step. The ligation rates observed with the different divalent metal ion catalysts (Mn2+ > Mg2+ > Ca2+ > Sr2+ = Ba2+) vary inversely with the pK(a) values of their bound water molecules. The pH profile and these relative ligation rates suggest a mechanism in which a metal-bound hydroxide ion located near the ligation junction promotes catalysis, most likely by deprotonation of the hydroxyl nucleophile. The effects of changing either the leaving group or the attacking hydroxyl, together with the large Delta S-double dagger value for oligonucleotide ligation (about -20 eu), are consistent with an associative transition state.