tRNase Z catalysis and conserved residues on the carboxy side of the His cluster
tRNase Z catalysis and conserved residues on the carboxy side of the His cluster
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DOI：
10.1021/bi700578v
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发表时间：
2007-08-21
期刊：
影响因子：
2.9
通讯作者：
Levinger, Louis
中科院分区：
文献类型：
--
作者：
Karkashon, Shay;Hopkinson, Angela;Levinger, Louis
tRNA作为前体转录，并在一系列所需的反应中加工，导致氨酰化和翻译。3 '-末端尾部可以通过前tRNA加工核酸内切酶tRNase Z去除，tRNase Z是金属依赖性水解酶的β-内酰胺酶超家族的古老保守成员。该家族的标志性序列、His结构域（HxHxDH、Motif II）以及Motif III和V中的组氨酸和Motif IV中的天冬氨酸为两个二价金属离子的配位贡献了七条侧链。我们以前研究了在基序II和PxKxRN环和基序I的基序II的氨基侧的取代对催化的影响。在此，我们提出了基序III，IV，HEAT和HST环，和基序V内的基序II的羧基侧上的取代的影响。基序IV天冬氨酸的取代降低催化效率超过10，000倍。基序III、V和HST环中的组氨酸在功能上也很重要。引人注目的是，在HEAT环中用Ala替换Glu会使效率降低约1000倍。这种Glu侧链相对于HST环中His的接近性和取向以及两个残基对于催化的重要性表明它们在反应的最后阶段作为质子转移中的二人组起作用，这是tRNase Z类RNA内切核酸酶的特征。
tRNAs are transcribed as precursors and processed in a series of required reactions leading to aminoacylation and translation. The 3'-end trailer can be removed by the pre-tRNA processing endonuclease tRNase Z, an ancient, conserved member of the beta-lactamase superfamily of metal-dependent hydrolases. The signature sequence of this family, the His domain (HxHxDH, Motif II), and histidines in Motifs III and V and aspartate in Motif IV contribute seven side chains for the coordination of two divalent metal ions. We previously investigated the effects on catalysis of substitutions in Motif II and in the PxKxRN loop and Motif I on the amino side of Motif II. Herein, we present the effects of substitutions on the carboxy side of Motif II within Motifs III, IV, the HEAT and HST loops, and Motif V. Substitution of the Motif IV aspartate reduces catalytic efficiency more than 10,000-fold. Histidines in Motif III, V, and the HST loop are also functionally important. Strikingly, replacement of Glu in the HEAT loop with Ala reduces efficiency by similar to 1000-fold. Proximity and orientation of this Glu side chain relative to His in the HST loop and the importance of both residues for catalysis suggest that they function as a duo in proton transfer at the final stage of reaction, characteristic of the tRNase Z class of RNA endonucleases.