A lead-dependent DNAzyme with a two-step mechanism
A lead-dependent DNAzyme with a two-step mechanism
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DOI：
10.1021/bi027332w
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发表时间：
2003-06-17
期刊：
影响因子：
2.9
通讯作者：
Lu, Y
中科院分区：
文献类型：
--
作者：
Brown, AK;Li, J;Lu, Y
详细的生物化学和机制的研究，在体外选定的8-17 DNA酶的变体。尽管8-17 DNAzyme基序已经通过在三种不同条件下的体外选择获得，所述条件包括10 mM Mg 2 +（称为8-17），0.5 mM Mg 2 +/50 mM组氨酸（称为Mg 5），或100 μ M Zn 2 +（称为17 E），所有变体都显示出与Pb 2+最活跃（8-17：k（obs）类似于0.5 min（-1）; Mg 5：k（obs）类似于2 min（-1）; 17 E：k（obs）类似于1 min（-1），200 μ M Pb 2+，pH 5.0）。对于8-17 DNA酶的17 E变体，单转换速率常数遵循Pb 2+远大于Zn 2+远大于Mn 2+近似于Co 2 + > Ni 2 + > Mg 2+近似于Ca 2 + > Sr 2+近似于Ba 2+的顺序。在13.5 μ M Pb ~（2+）、0.97 mM Zn ~（2+）或10.5 mM Mg ~（2+）时，催化速率为半最大，表明DNA酶的金属结合亲和力顺序为Pb ~（2+）> Zn ~（2+）> Mg ~（2+）。Pb ~（2+）依赖的活性随pH线性增加，log k（obs）对pH的斜率接近1，表明在反应的限速步骤中存在单一的去质子化。DNA酶的序列变异证实了G.T摆动对、两个环和插入茎在维持系统活性构象中的重要性。Mg 2+和Zn 2+仅催化酯交换反应，形成含有2 '，3'-环状磷酸酯的产物，而Pb 2+催化酯交换反应，随后水解2 '，3'-环状磷酸酯。虽然这种两步机制已被证明在蛋白质核糖核酸酶和前导酶RNAzyme中有效，但现在首次证明这种DNAzyme也可能使用相同的机制。因此，在所有类别的金属酶中观察到两步机制，并且这种8-17 DNAzyme为理解Pb 2+结合位点的结构及其在两步机制中的作用提供了简单、稳定且具有成本效益的模型系统。
A detailed biochemical and mechanistic study of in vitro selected variants of 8-17 DNAzymes is presented. Even though the 8-17 DNAzyme motif has been obtained through in vitro selection under three different conditions involving 10 mM Mg2+ (called 8-17), 0.5 mM Mg2+/50 mM histidine (called Mg5), or 100 muM Zn2+ (called 17E), all variants are shown to be the most active with Pb2+ (8-17: k(obs) similar to0.5 min(-1); Mg5: k(obs) similar to2 min(-1); 17E: k(obs) similar to1 min(-1) with 200 muM Pb2+ at pH 5.0). For the 17E variant of the 8-17 DNAzyme, the single-turnover rate constants followed the order of Pb2+ much greater than Zn2+ much greater than Mn2+ approximate to Co2+ > Ni2+ > Mg2+ approximate to Ca2+ > Sr2+ approximate to Ba2+. The catalytic rate is half-maximal at 13.5 muM Pb2+, 0.97 mM Zn2+, or 10.5 mM Mg2+, suggesting that the metal-binding affinity of the DNAzymes is in the order of Pb2+ > Zn2+ > Mg2+. The Pb2+-dependent activity increases linearly with pH and the slope of the plot of log k(obs) versus pH is similar to1, suggesting a single deprotonation in the rate-limiting step of the reaction. Sequence variations of the DNAzyme confirm the importance of the G.T wobble pair, the two loops and the intervening stem in maintaining the active conformation of the system. While Mg2+ and Zn2+ catalyze only a transesterification reaction with formation of a product containing a 2',3'-cyclic phosphate, Pb2+ catalyzes a transesterification reaction followed by hydrolysis of the 2',3'-cyclic phosphate. Although this two-step mechanism has shown to be operative in protein ribonucleases and in the leadzyme RNAzyme, it is now demonstrated for the first time that this DNAzyme may also use the same mechanism. Therefore, the two-step mechanism is observed in metalloenzymes of all classes, and this 8-17 DNAzyme provides a simple, stable, and cost-effective model system for understanding the structure of Pb2+-binding sites and their roles in the two-step mechanism.