Effect of alanine-293 replacement on the activity, ATP binding, and editing of Escherichia coli leucyl-tRNA synthetase

J. F. Chen, T. Li, E. D. Wang, Y. L. Wang

Research output: Contribution to journalArticlepeer-review

21 Scopus citations

Abstract

Leucyl-tRNA synthetase (LeuRS) is a class I aminoacyl-tRNA synthetase that catalyzes leucylation of tRNALeu. Several mutants in the CP1 domain of Escherichia coli LeuRS were obtained by introduction of restriction endonuclease sites into its gene, leuS. Of these mutants, only LeuRS-A293F had decreased activity (46%) compared to the native enzyme. To investigate the effect of A293 on enzyme function, A293 was mutated to Y, G, I, R, or D. The mutants were impaired in activity and editing function to varying extents. The decrease in Km values for three substrates showed that the binding of ATP to these mutants became much stronger. The inhibition of ATP binding to most of the mutants was also stronger. In particular, LeuRS-A293D had the lowest activity, the strongest ATP binding, and the most impaired editing function. A red shift of the fluorescence emission maximum of LeuRS-A293D indicated a less hydrophobic chromophore environment and a relatively more flexible dynamic conformation. The change in Tm of LeuRS-A293D was higher than that of all other substitutions. Evidence from sequence alignment and crystal structure of LeuRS from Thermus thermophilus shows that A293 was conserved as R (K) or A and is located at a small helix in the editing domain of the enzyme facing the active site. Hence, any amino acid substitution of A293 may affect the stability of the helix, which may lead to impaired editing function and aminoacylation activity and may be indirectly involved in ATP binding.

Original languageEnglish (US)
Pages (from-to)1144-1149
Number of pages6
JournalBiochemistry
Volume40
Issue number5
DOIs
StatePublished - Feb 6 2001
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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