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I 类氨酰-tRNA 合成酶的合成和编辑途径之间的动力学分配发生在前转移和后转移水解步骤。

Kinetic partitioning between synthetic and editing pathways in class I aminoacyl-tRNA synthetases occurs at both pre-transfer and post-transfer hydrolytic steps.

机构信息

Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, 10000 Zagreb, Croatia.

出版信息

J Biol Chem. 2012 Jul 20;287(30):25381-94. doi: 10.1074/jbc.M112.372151. Epub 2012 May 30.

Abstract

Comprehensive steady-state and transient kinetic studies of the synthetic and editing activities of Escherichia coli leucyl-tRNA synthetase (LeuRS) demonstrate that the enzyme depends almost entirely on post-transfer editing to endow the cell with specificity against incorporation of norvaline into protein. Among the three class I tRNA synthetases possessing a dedicated post-transfer editing domain (connective peptide 1; CP1 domain), LeuRS resembles valyl-tRNA synthetase in its reliance on post-transfer editing, whereas isoleucyl-tRNA synthetase differs in retaining a distinct tRNA-dependent synthetic site pre-transfer editing activity to clear noncognate amino acids before misacylation. Further characterization of the post-transfer editing activity in LeuRS by single-turnover kinetics demonstrates that the rate-limiting step is dissociation of deacylated tRNA and/or amino acid product and highlights the critical role of a conserved aspartate residue in mediating the first-order hydrolytic steps on the enzyme. Parallel analyses of adenylate and aminoacyl-tRNA formation reactions by wild-type and mutant LeuRS demonstrate that the efficiency of post-transfer editing is controlled by kinetic partitioning between hydrolysis and dissociation of misacylated tRNA and shows that trans editing after rebinding is a competent kinetic pathway. Together with prior analyses of isoleucyl-tRNA synthetase and valyl-tRNA synthetase, these experiments provide the basis for a comprehensive model of editing by class I tRNA synthetases, in which kinetic partitioning plays an essential role at both pre-transfer and post-transfer steps.

摘要

全面的稳态和瞬态动力学研究表明,大肠杆菌亮氨酰-tRNA 合成酶(LeuRS)的合成和编辑活性几乎完全依赖于转移后编辑,赋予细胞特异性以防止正缬氨酸掺入蛋白质。在具有专用转移后编辑结构域(连接肽 1;CP1 结构域)的三个 I 类 tRNA 合成酶中,LeuRS 类似于缬氨酰-tRNA 合成酶,依赖于转移后编辑,而异亮氨酰-tRNA 合成酶则不同,它保留了独特的 tRNA 依赖性合成位点预转移编辑活性,以在错氨酰化之前清除非同源氨基酸。通过单轮动力学对 LeuRS 转移后编辑活性的进一步表征表明,限速步骤是去酰化 tRNA 和/或氨基酸产物的解离,并突出了保守天冬氨酸残基在介导酶的一级水解步骤中的关键作用。野生型和突变型 LeuRS 的腺苷酸和氨酰-tRNA 形成反应的平行分析表明,转移后编辑的效率受水解和去酰化错误氨酰化 tRNA的解离之间的动力学分配控制,并表明再结合后的转编辑是一种有效的动力学途径。结合对异亮氨酰-tRNA 合成酶和缬氨酰-tRNA 合成酶的先前分析,这些实验为 I 类 tRNA 合成酶的编辑提供了全面的模型基础,其中动力学分配在预转移和转移后步骤中都起着至关重要的作用。

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