Kuimelis R G, McLaughlin L W
Department of Chemistry, Mekert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02167, USA.
Biochemistry. 1996 Apr 23;35(16):5308-17. doi: 10.1021/bi952994p.
An oligonucleotide substrate containing a 5'-bridging phosphorothioate linkage adjacent to a ribonucleotide has been used to investigate the cleavage mechanisms of the hammerhead ribozyme and to probe the catalytic role of the metal cofactor(s). Specifically, we tested the hypothesis that a second metal interacts with the 5'-leaving group to facilitate the cleavage event. To this end, we have examined the ribozyme-mediated cleavage activity of the phosphorothioate substrate at pH 7.5 with a series of divalent metal in both the presence and absence of the polycation spermine. The cleavage products are found to be the same as for the native sequence under a variety of reaction conditions. The influence of divalent metal ion concentration, temperature, and pH on the cleavage rate also has been examined for both the oxo linkage and the thio analogue. Spermine (but not spermidine or NaCl) is shown to support efficient cleavage of the thio analogue in the absence [5 mM ethylenediaminetetraacetic acid (EDTA)] of a divalent metal cofactor. The cleavage of the oxo linkage exhibits a solvent deuterium isotope effect of 3.6, but a similar effect is not observed with the thio analogue. The pseudo-first-order rate constants for cleavage of the thio analogue in the presence of 10 mM Mg2+ or Mn2+ at pH 7.5 are 65 and 82 x 10(-3) min-1, respectively. The native oxo linkage is cleaved at essentially the same rate as the thio analogue (35 and 97 x 10(-3) min-1 for Mg2+ and Mn2+, respectively). The absence of an appreciable thio effect and the lack of a preference for either Mg2+ or Mn2+ provides compelling evidence that the metal cofactor does not interact with the 5'-thioanion (or oxyanion) leaving group in the transition state. These rate comparisons additionally reveal the the departure of the 5'-leaving group is not the rate-limiting step of the cleavage reaction catalyzed by the hammerhead ribozyme.
一种在核糖核苷酸相邻位置含有5'-桥连硫代磷酸酯键的寡核苷酸底物已被用于研究锤头状核酶的切割机制，并探究金属辅因子的催化作用。具体而言，我们测试了一种假说，即第二种金属与5'-离去基团相互作用以促进切割事件。为此，我们在pH 7.5条件下，研究了在一系列二价金属存在和不存在聚阳离子精胺的情况下，硫代磷酸酯底物的核酶介导的切割活性。发现在各种反应条件下，切割产物与天然序列的相同。还研究了二价金属离子浓度、温度和pH对氧代连接和硫代类似物切割速率的影响。在不存在二价金属辅因子[5 mM乙二胺四乙酸（EDTA）]的情况下，精胺（而非亚精胺或NaCl）被证明能支持硫代类似物的有效切割。氧代连接的切割表现出3.6的溶剂氘同位素效应，但硫代类似物未观察到类似效应。在pH 7.5条件下，10 mM Mg2+或Mn2+存在时硫代类似物切割的伪一级速率常数分别为65和82×10(-3) min-1。天然氧代连接的切割速率与硫代类似物基本相同（Mg2+和Mn2+分别为35和97×10(-3) min-1）。硫代效应不明显以及对Mg2+或Mn2+均无偏好，提供了令人信服的证据，表明金属辅因子在过渡态下不与5'-硫阴离子（或氧阴离子）离去基团相互作用。这些速率比较还揭示了5'-离去基团的离去不是锤头状核酶催化的切割反应的限速步骤。
