Murray J B, Adams C J, Arnold J R, Stockley P G
Department of Genetics, University of Leeds, UK.
Biochem J. 1995 Oct 15;311 ( Pt 2)(Pt 2):487-94. doi: 10.1042/bj3110487.
We report details of the synthesis and characterization of oligoribonucleotides containing 4-thiouridine or 2-pyrimidinone ribonucleoside (4HC). We have used these probes to examine the roles of the conserved pyrimidines in the central core of the hammerhead ribozyme. The effects on catalysis of singly-substituted hammerhead ribozyme and substrate strands were quantified in multiple-turnover reactions. Various effects were observed on kcat. and Km, with up to a 7-fold decrease and a 3-fold increase respectively. For substitutions with 4HC at positions 3 or 17, catalytic activity in single turnover reactions can be increased up to 8-fold equivalent to 40% of wild-type activity, by increasing the concentration of the Mg2+ cofactor, implying that these substitutions had a deleterious effect on Mg2+ binding. Calculations of the change in the apparent free energy of binding for variants at positions 3, 4 or 17 are each consistent with deletion of a single hydrogen-bond to an uncharged group in the ribozyme. The cytidine 5' to the scissile phosphate had not previously been thought to play a direct role in catalysis, however, removal of the exocyclic amino group decreased kcat. 4-fold. Recently, the crystal structures of a hammerhead ribozyme bound to either a non-cleavable 2'-deoxy substrate strand or a ribo-substrate strand have been reported. The kinetic properties of the variants described here are consistent with several key interactions seen in the crystals, in particular they provide experimental support for the assignment of the proposed catalytically active magnesium ion-binding site.
我们报告了含4-硫尿苷或2-嘧啶酮核糖核苷（4HC）的寡核糖核苷酸的合成及表征细节。我们使用这些探针研究了锤头状核酶中心核心中保守嘧啶的作用。在多轮反应中对单取代锤头状核酶和底物链的催化作用进行了定量。观察到对催化常数（kcat.）和米氏常数（Km）有各种影响，分别最多降低7倍和增加3倍。对于在3或17位用4HC取代的情况，通过增加镁离子辅因子的浓度，单轮反应中的催化活性可提高至8倍，相当于野生型活性的40%，这意味着这些取代对镁离子结合有有害影响。对3、4或17位变体结合表观自由能变化的计算均与核酶中与一个不带电荷基团的单个氢键缺失一致。之前认为位于可切割磷酸基团5'端的胞嘧啶在催化中不直接起作用，然而，去除其环外氨基会使催化常数降低4倍。最近，已报道了与不可切割的2'-脱氧底物链或核糖底物链结合的锤头状核酶的晶体结构。此处描述的变体的动力学性质与晶体中观察到的几个关键相互作用一致，特别是它们为所提出的催化活性镁离子结合位点的归属提供了实验支持。
