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, tRNA(Gln) and ATP bind with dissociation constants of 460, 0.22 and 180 microM, 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/tRNA(Gln) crystal structure indicates that no major change in shape and size occurs upon tRNA(Gln) 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 tRNA(Gln) 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.
Bhattacharyya
T
T
Department of Biophysics, Bose Institute, Calcutta, India.
Bhattacharyya
A
A
Roy
S
S
D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D005456:Fluorescent Dyes; D005975:Glutamate-tRNA Ligase / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism; D008301:Maleimides / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D011487:Protein Conformation; D012351:RNA, Transfer, Gln / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D008301:Maleimides; D010710:Phosphates; D012351:RNA, Transfer, Gln; D005973:Glutamine; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; C003944:N-(3-pyrene)maleimide; D005975:Glutamate-tRNA Ligase