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首页> 外文期刊>Biochemistry >The peptide bond between E292-A293 of Escherichia coli leucyl-tRNA synthetase is essential for its activity.
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The peptide bond between E292-A293 of Escherichia coli leucyl-tRNA synthetase is essential for its activity.

机译：大肠杆菌亮氨酰tRNA合成酶的E292-A293之间的肽键对其活性至关重要。

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摘要

Escherichia coli leucyl-tRNA synthetase (LeuRS) is a class I aminoacyl-tRNA synthetase that contains a large connecting polypeptide (CP1) inserted into its nucleotide binding fold, or active site. In this study, purified leucyl-tRNA synthetase was found to be cleaved between E292 and A293 in its CP1 domain. SDS-PAGE analysis showed peptides of 63 and 34 kDa in addition to the native 97.3 kDa synthetase. By internal complementation, the two peptides could form a 97.3 kDa complex similar to the native LeuRS. This complex could support the ATP approximately PP(i) exchange activity of LeuRS, but could not complement for aminoacylation. To study the function of the region around the bond of E292 and A293, four pairs of peptides resulting from different cleavage sites in CP1 were reconstituted in vivo. With the exception of the enzyme assembled from the E292-A293 cleavage site, all the reassembled LeuRSs catalyzed the aminoacylation of tRNA(Leu). Although the E292-A293-cleaved LeuRS could not catalyze aminoacylation, fluorescence titration revealed that its tRNA binding ability was almost identical to that of wild-type LeuRS. These results suggest that the region around E292-A293 may be responsible for maintaining the proper conformation of LeuRS required for the tRNA charging activity.
机译：大肠杆菌亮氨酰-tRNA合成酶（LeuRS）是I类氨酰基-tRNA合成酶，其中包含一个大的连接多肽（CP1），插入其核苷酸结合倍数或活性位点。在这项研究中，发现纯化的亮氨酰-tRNA合成酶在其CP1域的E292和A293之间被切割。 SDS-PAGE分析显示，除天然97.3 kDa合成酶外，还有63和34 kDa的肽。通过内部互补，这两种肽可以形成类似于天然LeuRS的97.3 kDa复合物。该复合物可以支持LeuRS的ATP近似PP（i）交换活性，但不能补充氨基酰化作用。为了研究E292和A293键周围区域的功能，在体内重组了由CP1中不同切割位点产生的四对肽。除了从E292-A293切割位点组装的酶外，所有重新组装的LeuRS都催化tRNA（Leu）的氨酰化。尽管E292-A293切割的LeuRS无法催化氨酰化，但荧光滴定显示其tRNA结合能力与野生型LeuRS几乎相同。这些结果表明，E292-A293周围的区域可能负责维持tRNA充电活性所需的LeuRS的正确构象。

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