Kinetic and thermodynamic characterization of the RNA-cleaving 8-17 deoxyribozyme
Kinetic and thermodynamic characterization of the RNA-cleaving 8-17 deoxyribozyme
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DOI：
10.1093/nar/gkh250
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发表时间：
2004-02-01
影响因子：
14.9
通讯作者：
Peracchi, A
中科院分区：
文献类型：
--
作者：
Bonaccio, M;Credali, A;Peracchi, A
8-17 脱氧核酶是一种具有重要应用价值的小型 DNA 催化剂。我们分析了性能良好的8-17构建体的动力学特征，并确定了几种反应条件对这些特征的影响，为进一步探索脱氧核酶机制提供了基础。 8-17 结合底物的速率常数比短互补寡核苷酸退火的典型速率常数低 10 倍。观察到的底物结合自由能表明接近+7 kcal/mol 的能量损失可归因于脱氧核酶核心。底物裂解需要二价金属离子辅助因子，并且活性对 Mg2+、Ca2+ 或 Mn2+ 浓度的依赖性表明存在用于激活离子的单一低特异性结合位点。活化效率与离子辅助因子的路易斯酸度相关，与金属辅助的活性 2'-羟基去质子化相容。然而，不能排除金属离子的替代作用，因为那些更强的路易斯酸离子也能够与配体（例如磷酸盐氧）形成更强的相互作用。 8-17 反应的表观活化焓接近于氢氧化物催化和锤头核酶催化的 RNA 切割观察到的值。
The 8-17 deoxyribozyme is a small DNA catalyst of significant applicative interest. We have analyzed the kinetic features of a well behaved 8-17 construct and determined the influence of several reaction conditions on such features, providing a basis for further exploration of the deoxyribozyme mechanism. The 8-17 bound its substrate with a rate constant similar to10-fold lower than those typical for the annealing of short complementary oligonucleotides. The observed free energy of substrate binding indicates that an energetic penalty near to +7 kcal/mol is attributable to the deoxyribozyme core. Substrate cleavage required divalent metal ion cofactors, and the dependence of activity on the concentration of Mg2+, Ca2+ or Mn2+ suggests the occurrence of a single, low-specificity binding site for activating ions. The efficiency of activation correlated with the Lewis acidity of the ion cofactor, compatible with a metal-assisted deprotonation of the reactive 2'-hydroxyl group. However, alternative roles of the metal ions cannot be excluded, because those ions that are stronger Lewis acids are also capable of forming stronger interactions with ligands such as the phosphate oxygens. The apparent enthalpy of activation for the 8-17 reaction was close to the values observed for hydroxide-catalyzed and hammerhead ribozyme-catalyzed RNA cleavage.