Robert B. Loftfield

[48] Kinetic techniques for the investigation of amino acid: tRNA ligases (aminoacyl-tRNA synthetases, amino acid activating enzymes)☆

Publisher Summary This chapter describes four kinetic assays for investigating amino acid:tRNA ligases: adenosine triphosphate:pyrophosphate (ATP:PPi) exchange assay, the hydroxamate assay, tRNA esterification assay, and adenosine monophosphate (AMP) production assay. The ATP:PPI exchange assay has been widely and profitably used to measure Kis for a variety of amino acid derivatives or ATP analogs that do not catalyze the ATP:PPI exchange. The hydroxamate technique is useful for the reaction of all the neutral amino acids. tRNA esterification method has been modified slightly to give best conditions for doing kinetic studies on purified ligases. This assay has the advantage of being very specific for a particular enzyme, amino acid, and tRNA. AMP production assay is responsive to any ATPases and to all kinds of ATP (AMP) ligases. In addition to the kinetic methods, some other techniques that have been especially useful in studying the kinetic properties of these enzymes are (1) Single-pass kinetics, (2) kinetic determinations of the isolated step of association and the membrane filter technique, (3) equilibrium and nonequilibrium dialysis techniques, and (4) possibly the most powerful tool, fluorescence analysis.