Abstract
For tRNA-dependent protein biosynthesis, amino acids are first activated by aminoacyl-tRNA synthetases (aaRSs) yielding the reaction intermediates aminoacyl-AMP (aa-AMP). Stable analogues of aa-AMP, such as aminoacyl-sulfamoyl-adenosines, inhibit their cognate aaRSs. Glutamyl-sulfamoyl-adenosine (Glu-AMS) is the best known inhibitor of Escherichia coli glutamyl-tRNA synthetase (GluRS). Thermodynamic parameters of the interactions between Glu-AMS and E. coli GluRS were measured in the presence and in the absence of tRNA by isothermal titration microcalorimetry. A significant entropic contribution for the interactions between Glu-AMS and GluRS in the absence of tRNA or in the presence of the cognate tRNAGlu or of the non-cognate tRNAPhe is indicated by the negative values of –TΔSb, and by the negative value of ΔCp. On the other hand, the large negative enthalpy is the dominant contribution to ΔGb in the absence of tRNA. The affinity of GluRS for Glu-AMS is not altered in the presence of the non-cognate tRNAPhe, but the dissociation constant K d is decreased 50-fold in the presence of tRNAGlu; this result is consistent with molecular dynamics results indicating the presence of an H-bond between Glu-AMS and the 3’-OH oxygen of the 3’-terminal ribose of tRNAGlu in the Glu-AMS•GluRS•tRNAGlu complex. Glu-AMS being a very close structural analogue of Glu-AMP, its weak binding to free GluRS suggests that the unstable Glu-AMP reaction intermediate binds weakly to GluRS; these results could explain why all the known GluRSs evolved to activate glutamate only in the presence of tRNAGlu, the coupling of glutamate activation to its transfer to tRNA preventing unproductive cleavage of ATP.
Highlights
Aminoacyl-tRNA synthetases play a front-line role in protein biosynthesis; they are responsible for the attachment of specific amino acids to their cognate tRNAs
This two-step reaction begins with the amino acid activation by condensation with an ATP molecule, creating an aminoacyl-adenylate which reacts on the aminoacyltRNA synthetases (aaRSs) with the 3’-terminal adenosine of the tRNA’s acceptor stem, giving the aminoacyl-tRNA which participates in protein biosynthesis on the ribosome
A substantial entropic contribution for the interactions between Glu-AMS and glutamyl-tRNA synthetase (GluRS) in the absence of tRNA or in the presence of the cognate tRNAGlu or of the non-cognate tRNAPhe is indicated by the negative values of-TΔSb under these three conditions (Fig 2)
Summary
Aminoacyl-tRNA synthetases (aaRS) play a front-line role in protein biosynthesis; they are responsible for the attachment of specific amino acids to their cognate tRNAs. Several types of stable analogues of aa-AMP inhibit aaRSs (reviewed by Chênevert et al, 2003, and by Finn and Tao, 2005) [4,5]. Glutamyl-sulfamoyl-adenosine (Glu-AMS) (Fig 1) is a competitive inhibitor of Escherichia coli glutamyl-tRNA synthetase (GluRS) with a Ki of 2.8 nM [6] and is 25 times less efficient against murine hepatic GluRS. This result suggests that the structures of the active sites of bacterial and mammalian GluRSs differ significantly, and indicates that Glu-AMS derivatives with bactericidal properties and low toxicity for humans could be developed
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