Probing the Catalytic Triad of an Archaeal RNA Splicing Endonuclease
Probing the Catalytic Triad of an Archaeal RNA Splicing Endonuclease
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DOI：
10.1021/bi801141q
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发表时间：
2008-12-23
期刊：
影响因子：
2.9
通讯作者：
Li, Hong
中科院分区：
文献类型：
--
作者：
Calvin, Kate;Xue, Song;Li, Hong
在四种已知的内含子去除机制中，据说有三种是由RNA分子催化的。在第四种机制中，蛋白质核酸内切酶从核tRNA和所有古细菌RNA中去除内含子。剪接核酸内切酶的三个严格保守的残基，组氨酸，赖氨酸和酪氨酸，被预测以类似于核糖核酸酶A的催化三联体的方式催化内含子切割反应。单营业额动力学参数获得的野生型酶和两个三联体突变体。组氨酸突变为丙氨酸产生至少相似于28倍的降低;酪氨酸突变为苯丙氨酸产生至少相似于7倍的活性降低，而组氨酸和酪氨酸双突变消除切割。赖氨酸到谷氨酸的单突变在不存在二价金属的情况下废除了RNA切割活性，但在特定二价金属的存在下保持了相当水平的活性。这些数据支持已经提出的催化三联体的重要功能作用，并提出了一个有趣的假设，其中剪接核酸内切酶是从RNA到RNP世界过渡的中间体。
Among the four known mechanisms of intron removal, three are reputedly catalyzed by RNA molecules. In the fourth mechanism, a protein endonuclease removes introns from nuclear tRNA and all archaeal RNAs. Three strictly conserved residues of the splicing endonuclease, a histidine, a lysine, and a tyrosine, were predicted to catalyze the intron cleavage reaction in a manner similar to that of the catalytic triad of ribonuclease A. Single-turnover kinetic parameters were obtained for the wild-type enzyme and two triad mutants. Mutation of histidine to alanine produced an at least similar to 28-fold reduction; mutation of tyrosine to phenylalanine produced an at least similar to 7-fold reduction in activity, while a histidine and tyrosine double mutation abolished cleavage. The single mutation of lysine to glutamic acid abolished RNA cleavage activity in the absence of a divalent metal but maintained a substantial level of activity in the presence of specific divalent metals. These data support important functional roles already proposed for the catalytic triad and suggest an intriguing hypothesis in which the splicing endonuclease is an intermediate in the transition from the RNA to the RNP world.