A fluorescence spectroscopic study of glutaminyl‐tRNA synthetase from Escherichia coli and its implications for the enzyme mechanism

Abstract

Interaction between Escherichia coli glutaminyl-tRNA synthetase (GlnRS) and its substrates have been studied by fluorescence quenching. In the absence of other substrates, glutamine, tRNAG'" and ATP bind with dissociation constants of 460, 0.22 and 180 pM, 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'-(methy1)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/tRNAG1" crystal structure indicates that no major change in shape and size occurs upon tRNAG'" binding to GlnRS. S,S'-Bis(8-anilino-l-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 tRNAG'" 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.

A cognate tRNA specific conformational change in glutaminyl‐tRNA synthetase and its implication for specificity

“…Thus, we have measured tRNAG'" binding to GlnRS by quenching of the tryptophan fluorescence. Previously we have shown that binding of tRNAG1" leads to significant quenching of tryptophan fluorescence (Bhattacharyya et al, 1991). to the GlnRS/ATP complex reported here is in contrast to approximately 300-fold difference in binding constant in the absence of ATP reported by Weygand-Durasevic et al (1993), suggesting a strong ATP effect and possibly ordered substrate binding.…”

“…Enzyme assays are also described in the same article. The protein preparation showed a single band on SDS polyacrylamide gel and had specific activity comparable to those reported in Bhattacharyya et al (1991).…”

“…Glutaminyl-tRNA synthetase was purified according to Hoben et al (1982) with some minor modifications as described by Bhattacharyya et al (1991). Enzyme assays are also described in the same article.…”

“…Cells harboring pRS3 were grown according to the method described by Perona et al (1988), and tRNAG'" was purified from harvested cells using the methodology described in Bhattacharyya et al (1991). The specific activities of around 2 nmol/Azho units were obtained.…”

Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases

“…Alternatively, the aaRS can be chemically modified by derivatization with an extrinsic probe that is sensitive to local chemistry, thereby serving as a reporter of local changes in structure. The extrinsic probes employed include those that interact with the protein non-covalently, such as 5′,5′-bis(8-anilino-1-naphthalene sulfonate [67] and AF-tetrafluorophenylester [68], or those that can be covalently linked to exposed cysteines by maleimide chemistry [69]. Alternatively, if the labeling of the protein is not feasible, methods have been described for the extrinsic labeling of the 5′-terminus of the tRNA with a fluorophore [70,71].…”