Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases

Abstract

The accuracy of protein synthesis relies on the ability of aminoacyl-tRNA synthetases (aaRSs) to discriminate among true and near cognate substrates. To date, analysis of aaRSs function, including identification of residues of aaRS participating in amino acid and tRNA discrimination, has largely relied on the steady state kinetic pyrophosphate exchange and aminoacylation assays. Pre-steady state kinetic studies investigating a more limited set of aaRS systems have also been undertaken to assess the energetic contributions of individual enzyme-substrate interactions, particularly in the adenylation half reaction. More recently, a renewed interest in the use of rapid kinetics approaches for aaRSs has led to their application to several new aaRS systems, resulting in the identification of mechanistic differences that distinguish the two structurally distinct aaRS classes. Here, we review the techniques for thermodynamic and kinetic analysis of aaRS function. Following a brief survey of methods for the preparation of materials and for steady state kinetic analysis, this review will describe pre-steady state kinetic methods employing rapid quench and stopped-flow fluorescence for analysis of the activation and aminoacyl transfer reactions. Application of these methods to any aaRS system allows the investigator to derive detailed kinetic mechanisms for the activation and aminoacyl transfer reactions, permitting issues of substrate specificity, stereochemical mechanism, and inhibitor interaction to be addressed in a rigorous and quantitative fashion.

Homologs of aminoacyl-tRNA synthetases acylate carrier proteins and provide a link between ribosomal and nonribosomal peptide synthesis

“…This finding was quite puzzling, since all three homologs seemed to be properly folded as evaluated by circular dichroism spectroscopy and have dimeric structure as shown by size-exclusion chromatography. The activation of all 20 standard amino acids by the aSerRS homologs was therefore tested by a complementary method, the amino acid-depended ATP-pyrophosphate exchange assay (16). The results confirmed that only A. tumefaciens homolog is capable of serine activation, but revealed that alanine and glycine are also its substrates (Fig.…”

“…ATP-PP i exchange assay was performed at 30°C in 50 mM TrisHCl pH 7.5, 150 mM KCl, 0.2 mg∕mL BSA, 4 mM DTT, 20 mM MgCl 2 , 4 mM ATP, and 1 mM ½ 32 P − PP i , and varying concentrations of amino acids. Reaction was quenched, products were separated on polyethylenimine-cellulose TLC plates and quantified by phosphorimaging (16).…”

“…In spite of weak, but evident sequence similarity to aSerRSs, only A. tumefaciens homolog was able to activate serine in the standard activesite titration assay (16), while the other two homologs from B. japonicum appeared catalytically inactive. This finding was quite puzzling, since all three homologs seemed to be properly folded as evaluated by circular dichroism spectroscopy and have dimeric structure as shown by size-exclusion chromatography.…”

Leucyl-tRNA synthetase editing domain functions as a molecular rheostat to control codon ambiguity in Mycoplasma pathogens

“…Because 2′dA-tRNA and ddA-tRNA are missing the functional 2′ hydroxyl group that is necessary for aminoacylation to occur, AMP formation in these reactions would reflect cycles of amino acid activation and hydrolysis that are representative of pretransfer editing (31,32).…”

Plant tumour biocontrol agent employs a tRNA-dependent mechanism to inhibit leucyl-tRNA synthetase

“…The reaction was carried out at 28 1C and 5 ml time point aliquots collected. The assay was performed in triplicate and the active enzyme concentration was determined by measuring the amplitude of the burst phase of Leu-AMP formation 24,25 .…”