Partitioning of tRNA-dependent Editing between Pre- and Post-transfer Pathways in Class I Aminoacyl-tRNA Synthetases
Partitioning of tRNA-dependent Editing between Pre- and Post-transfer Pathways in Class I Aminoacyl-tRNA Synthetases
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DOI：
10.1074/jbc.m110.133553
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发表时间：
2010-07-30
影响因子：
4.8
通讯作者：
Gruic-Sovulj, Ita
中科院分区：
文献类型：
--
作者：
Dulic, Morana;Cvetesic, Nevena;Gruic-Sovulj, Ita
水解编辑活性存在于氨基酰基- trna合成酶中，在合成反应中具有较低的氨基酸辨别能力。在I类编辑酶中，misacylated tRNA的转移后水解发生在插入催化Rossmann折叠的空间分离结构域中，但misacylated氨基酸的转移前水解的位置和机制尚不确定。在这里，我们使用新的动力学方法来区分大肠杆菌异亮氨酸- trna合成酶（IleRS）的三种预转移编辑模型。我们证明了非同源戊酸腺苷酸的trna依赖性水解在很大程度上对酶编辑结构域的突变不敏感，并且释放后的非催化水解太慢，无法解释观察到的清除率。对氨基酸转移到tRNA的微观速率常数和相关valyl-tRNA合成酶（ValRS）的测量进一步表明，在IleRS中，转移前编辑是一种存在于合成活性位点的酶催化活性。在该模型中，转移前和转移后编辑途径之间的平衡由非同源氨基酸酰腺苷酸的动力学分配控制。在IleRS中，非同源中间体的水解和转移的速率常数大致相等，而在ValRS中，向tRNA的转移比水解快200倍。因此，ValRS的编辑几乎完全通过编辑域的转移后水解发生，而在IleRS中，转移前和转移后的编辑都很重要。在这两种酶中，同源和非同源的氨基酸酰腺苷酸转移到tRNA的速率相似，这与编辑DNA聚合酶形成了显著对比。
Hydrolytic editing activities are present in aminoacyl-tRNA synthetases possessing reduced amino acid discrimination in the synthetic reactions. Post-transfer hydrolysis of misacylated tRNA in class I editing enzymes occurs in a spatially separate domain inserted into the catalytic Rossmann fold, but the location and mechanisms of pre-transfer hydrolysis of misactivated amino acids have been uncertain. Here, we use novel kinetic approaches to distinguish among three models for pre-transfer editing by Escherichia coli isoleucyl-tRNA synthetase (IleRS). We demonstrate that tRNA-dependent hydrolysis of noncognate valyl-adenylate by IleRS is largely insensitive to mutations in the editing domain of the enzyme and that noncatalytic hydrolysis after release is too slow to account for the observed rate of clearing. Measurements of the microscopic rate constants for amino acid transfer to tRNA in IleRS and the related valyl-tRNA synthetase (ValRS) further suggest that pre-transfer editing in IleRS is an enzyme-catalyzed activity residing in the synthetic active site. In this model, the balance between pre-transfer and post-transfer editing pathways is controlled by kinetic partitioning of the noncognate aminoacyl-adenylate. Rate constants for hydrolysis and transfer of a noncognate intermediate are roughly equal in IleRS, whereas in ValRS transfer to tRNA is 200-fold faster than hydrolysis. In consequence, editing by ValRS occurs nearly exclusively by post-transfer hydrolysis in the editing domain, whereas in IleRS both pre- and post-transfer editing are important. In both enzymes, the rates of amino acid transfer to tRNA are similar for cognate and noncognate aminoacyl-adenylates, providing a significant contrast with editing DNA polymerases.