The peptide bond between E292-A293 of Escherichia coli leucyl-tRNA synthetase is essential for its activity

Tong Li, Nini Guo, Xian Xia, En Duo Wang, Ying Lai Wang

Research output: Contribution to journalArticle

Abstract

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~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.

Original languageEnglish (US)
Pages (from-to)13063-13069
Number of pages7
JournalBiochemistry
Volume38
Issue number40
DOIs
StatePublished - Oct 5 1999
Externally publishedYes

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Amino Acyl-tRNA Synthetases
Escherichia coli
Peptides
Aminoacylation
Transfer RNA
Transfer RNA Aminoacylation
RNA, Transfer, Leu
Ligases
Titration
Conformations
Polyacrylamide Gel Electrophoresis
Catalytic Domain
Nucleotides
Adenosine Triphosphate
Fluorescence
Binding Sites
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

The peptide bond between E292-A293 of Escherichia coli leucyl-tRNA synthetase is essential for its activity. / Li, Tong; Guo, Nini; Xia, Xian; Wang, En Duo; Wang, Ying Lai.

In: Biochemistry, Vol. 38, No. 40, 05.10.1999, p. 13063-13069.

Research output: Contribution to journalArticle

Li, Tong ; Guo, Nini ; Xia, Xian ; Wang, En Duo ; Wang, Ying Lai. / The peptide bond between E292-A293 of Escherichia coli leucyl-tRNA synthetase is essential for its activity. In: Biochemistry. 1999 ; Vol. 38, No. 40. pp. 13063-13069.
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