Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases.
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.
Francklyn
Christopher S
CS
Department of Biochemistry, University of Vermont, Health Sciences Complex, 89 Beaumont Avenue, Burlington, VT 05405, USA. Christopher.Francklyn@uvm.edu
First
Eric A
EA
Perona
John J
JJ
Hou
Ya-Ming
YM
D000604:Amino Acyl-tRNA Synthetases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001671:Biochemistry / Q000379:methods; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D046249:Transfer RNA Aminoacylation; D014764:Viral Proteins / Q000378:metabolism
D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D000604:Amino Acyl-tRNA Synthetases