Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, 10000 Zagreb, Croatia.
J Biol Chem. 2012 Jul 20;287(30):25381-94. doi: 10.1074/jbc.M112.372151. Epub 2012 May 30.
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.
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。