Santoro S W, Joyce G F
Department of Chemistry, The Skaggs Institute for Chemical Biology, La Jolla, California 92037, USA.
Biochemistry. 1998 Sep 22;37(38):13330-42. doi: 10.1021/bi9812221.
We previously reported the in vitro selection of a general-purpose RNA-cleaving DNA enzyme that exhibits a catalytic efficiency (kcat/KM) 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 approximately 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.
我们之前报道了一种通用型RNA切割DNA酶的体外筛选，该酶的催化效率（kcat/KM）超过任何其他已知的核酸酶[桑托罗，S. W.和乔伊斯，G. F.（1997年）《美国国家科学院院刊》94，4262 - 4266]。这种酶含有大约30个脱氧核苷酸，在模拟生理条件下几乎可以切割任何RNA底物，通过两个沃森-克里克结合结构域识别底物。在不同pH条件、二价金属辅因子的选择以及二价金属浓度下的切割动力学与一种化学机制一致，该机制涉及金属辅助使底物的2'-羟基去质子化，从而导致底物切割。动力学测量表明，该酶强烈倾向于切割底物而非连接两个切割产物，并且酶的催化效率受底物结合速率的限制。该酶表现出高度的底物特异性，能够区分在两个底物识别结构域中任何一个内含有单个碱基错配的RNA。通过对底物识别结构域进行适当设计，该酶展现出高底物序列特异性和选择性、高催化效率以及快速催化周转的强大组合。
