Tuschl T, Ng M M, Pieken W, Benseler F, Eckstein F
Max-Planck-Institut für experimentelle Medizin, Göttingen, Germany.
Biochemistry. 1993 Nov 2;32(43):11658-68. doi: 10.1021/bi00094a023.
The three guanosines of the central core of a hammerhead ribozyme were replaced by 2-aminopurine ribonucleoside, xanthosine, isoguanosine, inosine, and deoxyguanosine. These analogues were incorporated by automated solid-phase synthesis, with the exception of isoguanosine. This was introduced by ligating a donor, which carried the isoguanosine at its 5'-end, and an acceptor oligoribonucleotide by a T4 DNA ligase-catalyzed reaction. Most of these modifications lowered the rate constant of cleavage by the hammerhead ribozyme drastically. Inspection of the possible hydrogen-bonding interactions disturbed by these modifications suggests that there is no G12A9 or A13G8 mismatched base pair in the central region. Increasing the Mg2+ concentration from 10 to 50 mM did not enhance these rates appreciably. This makes it improbable that the guanosines, including their 2'-hydroxyl groups, are involved in the binding of the catalytically active Mg2+. Transition-state destabilizing energies of 0.6-4.7 kcal mol-1 suggest that essentially all guanosines are involved in a hydrogen-bonding network.
锤头状核酶中心核心的三个鸟苷被2-氨基嘌呤核糖核苷、黄苷、异鸟苷、肌苷和脱氧鸟苷取代。除异鸟苷外，这些类似物通过自动固相合成法掺入。异鸟苷是通过连接一个在其5'-末端携带异鸟苷的供体和一个受体寡核糖核苷酸，由T4 DNA连接酶催化反应引入的。这些修饰大多显著降低了锤头状核酶的切割速率常数。对受这些修饰干扰的可能氢键相互作用的检查表明，中心区域不存在G12A9或A13G8错配碱基对。将Mg2+浓度从10 mM增加到50 mM并没有明显提高这些速率。这使得包括其2'-羟基在内的鸟苷参与催化活性Mg2+的结合变得不太可能。0.6 - 4.7千卡/摩尔的过渡态去稳定能表明，基本上所有的鸟苷都参与了氢键网络。
