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

Leucyl-tRNA synthetase (LeuRS) is a class I aminoacyl-tRNA synthetase that catalyzes leucylation of tRNA(Leu). Several mutants in the CP1 domain of Es cherichia coli LeuRS were obtained by introduction of restriction endonucle ase sites into its gene, leuS. Of these mutants, only LeuRS-A293F had decre ased activity (46%) compared to the native enzyme. To investigate the effec t of A293 on enzyme function, A293 was mutated to Y, G, I, R, or D. The mut ants were impaired in activity and editing function to varying extents. The decrease in K-m values for three substrates showed that the binding of ATP to these mutants became much stronger. The inhibition of ATP binding to mo st of the mutants was also stronger. In particular, LeuRS-A293D had the low est activity, the strongest ATP binding, and the most impaired editing func tion. A red shift of the fluorescence emission maximum of LeuRS-A293D indic ated a less hydrophobic chromophore environment and a relatively more flexi ble dynamic conformation. The change in T-m of LeuRS-A293D was higher than that of all other substitutions. Evidence from sequence alignment and cryst al structure of LeuRS from Thermus thermophilus shows that A293 was conserv ed as R (K) or A and is located at a small helix in the editing domain of t he enzyme facing the active site. Hence, any amino acid substitution of A29 3 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.