3 research outputs found
Recommended from our members
A fluorescence spectroscopic study of glutaminyl-tRNA synthetase from Escherichia coli and its implications for the enzyme mechanism
Interaction between Escherichia coli glutaminyl-tRNA synthetase (GlnRS) and its substrates have been studied by fluorescence quenching. In the absence of other substrates, glutamine, tRNAGln and ATP bind with dissociation constants of 460, 0.22 and 180 µM, respectively. The presence of other substrates has either no effect or, at best a weak effect, on binding of ligands. Attempts to isolate enzyme-bound aminoacyl adenylate did not succeed. Binding of the phosphodiester, 5'-(methyl)adenosine monophosphate (MeAMP), to GlnRS was studied by fluorescence quenching and radioactive-ligand binding. tRNA also only has a weak effect on phosphodiester binding. Selectively pyrene-labeled GlnRS was used to obtain shape and size information for free GlnRS. A comparison with the GlnRS shape in the GlnRS/tRNAGln crystal structure indicates that no major change in shape and size occurs upon tRNAGln binding to GlnRS. 5,5'-Bis(8-anilino-1-naphthalene sulfonate) (bis-ANS), a non-covalent fluorescent probe, was also used to probe for conformational changes in GlnRS. This probe also indicated that no major conformational change occurs upon tRNAGln binding. We conclude that lack of tRNA-independent pyrophosphate-exchange activity in this enzyme is not a result of either lack of glutamine or ATP binding in the absence of tRNA, or formation of aminoacyl adenylate and slow release of pyrophosphate. A conformational change is implied upon tRNA binding, which promotes pyrophosphate exchange. Fluorescence studies indicate that this conformational change must be limited and local in nature
Recommended from our members
A cognate tRNA specific conformational change in glutaminyl-tRNA synthetase and its implication for specificity
Conformational changes that occur upon substrate binding are known to play crucial roles in the recognition and specific aminoacylation of cognate tRNA by glutaminyl-tRNA synthetase. In a previous study we had shown that glutaminyl-tRNA synthetase labeled selectively in a nonessential sulfhydryl residue by an environment sensitive probe, acrylodan, monitors many of the conformational changes that occur upon substrate binding. In this article we have shown that the conformational change that occurs upon tRNAGln binding to glnRS/ATP complex is absent in a noncognate tRNA tRNAGlu-glnRS/ATP complex. CD spectroscopy indicates that this cognate tRNAGln-induced conformational change may involve only a small change in secondary structure. The Van't Hoff plot of cognate and noncognate tRNA binding in the presence of ATP is similar, suggesting similar modes of interaction. It was concluded that the cognate tRNA induces a local conformational change in the synthetase that may be one of the critical elements that causes enhanced aminoacylation of the cognate tRNA over the noncognate ones
Recommended from our members
A fluorescence spectroscopic study of substrate-induced conformational changes in glutaminyl-tRNA synthetase
Glutaminyl-tRNA synthetase from Escherichia coli is a member of a subgroup of aminoacyl-tRNA synthetases that do not catalyze ATP-PPi exchange in the absence of the cognate tRNA. Such behavior suggests conformational changes upon substrate binding. Two different fluorescent probes, pyrenylmaleimide and acrylodan, were used to specifically label a nonessential sulfhydryl group of GlnRS. Conformational changes induced by substrates were studied using glutaminyl-tRNA synthetase labeled with these two environment-sensitive probes. ATP was shown to cause a significant conformational change that alters the mode of binding to tRNA(Gln) to GlnRS. The alteration of the salt sensitivity pattern of tRNA(Gln) binding to GlnRS by ATP supports this. Binding of tRNA(Gln) causes a conformational change that may be different in nature for the ATP/GlnRS complex and free GlnRS. Hydrodynamic parameters deduced from fluorescence polarization studies and the use of a noncovalent probe indicate that the ATP-induced conformational change may not be global in character