Mechanism and utility of an RNA-cleaving DNA enzyme
Mechanism and utility of an RNA-cleaving DNA enzyme
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DOI：
10.1021/bi9812221
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发表时间：
1998-09-22
期刊：
影响因子：
2.9
通讯作者：
Joyce, GF
中科院分区：
文献类型：
--
作者：
Santoro, SW;Joyce, GF
我们之前报道了一种通用 RNA 切割 DNA 酶的体外选择，其催化效率 (k(cat)/K-M) 超过任何其他已知的核酸酶 [Santoro, S. W. 和 Joyce, G. F. (1997) Proc.国家。阿卡德。科学。美国 94, 4262-4266]。这种酶含有类似 30 个脱氧核苷酸，在模拟生理条件下可以切割几乎任何 RNA 底物，通过两个 Watson-Crick 结合域识别底物。在不同pH、二价金属辅助因子的选择和二价金属浓度的条件下的裂解动力学与涉及底物2'-羟基的金属辅助去质子化、导致底物裂解的化学机制一致。动力学测量表明，与两种裂解产物的连接相比，该酶强烈倾向于底物的裂解，并且该酶的催化效率受到底物结合速率的限制。该酶表现出高水平的底物特异性，可区分两个底物识别域中任一区域内含有单碱基错配的 RNA。通过适当设计底物识别结构域，该酶表现出高底物序列特异性和选择性、高催化效率和快速催化周转的有效组合。
We previously reported the in vitro selection of a general-purpose RNA-cleaving DNA enzyme that exhibits a catalytic efficiency (k(cat)/K-M) exceeding that of any other known nucleic acid enzyme [Santoro, S. W. and Joyce, G. F. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 4262-4266]. This enzyme contains similar to 30 deoxynucleotides and can cleave almost any RNA substrate under simulated physiological conditions, recognizing the substrate through two Watson-Crick binding domains. The kinetics of cleavage under conditions of varying pH, choice of divalent metal cofactor, and divalent metal concentration are consistent with a chemical mechanism involving metal-assisted deprotonation of a 2'-hydroxyl of the substrate, leading to substrate cleavage. Kinetic measurements reveal that the enzyme strongly prefers cleavage of the substrate over ligation of the two cleavage products and that the enzyme's catalytic efficiency is limited by the rate of substrate binding. The enzyme displays a high level of substrate specificity, discriminating against RNAs that contain a single base mismatch within either of the two substrate-recognition domains. With appropriate design of the substrate-recognition domains, the enzyme exhibits a potent combination of high substrate sequence specificity and selectivity, high catalytic efficiency, and rapid catalytic turnover.