Kinetic characterization of amino acid activation by aminoacyl‐t<scp>RNA</scp> synthetases using radiolabelled γ‐[³²<scp>P</scp>]<scp>ATP</scp>

Aminoacyl-tRNA synthetases (AARSs) are fundamental enzymes that pair amino acids and tRNAs for protein synthesis. Aminoacylation occurs in two discrete steps. The amino acid is first activated by ATP, leading to an aminoacyl-adenylate intermediate and pyrophosphate (PP_i) formation. In a subsequent step, the aminoacyl moiety is transferred to the tRNA. Kinetic assays were developed to follow each of these steps independently, as well as cumulative two-step aminoacylation. The main advantage of following the activation step over two-step aminoacylation is that most AARSs can activate amino acids in the absence of the tRNA, the production of which is laborious. Hence, the activation step is often tested first in the kinetic analysis, including large screens exploring AARS-targeting inhibitors. Since the 1960s, the activation reaction has been routinely followed by the standard ATP/[³²P]PP_i exchange assay, which relies on the equilibrium exchange of radiolabel between PP_i and ATP using [³²P]PP_i as a labelled compound. However, this method became much less convenient when [³²P]PP_i was discontinued in 2022. As a solution, we developed a modified assay that uses easily attainable γ-[³²P]ATP as a labelled compound in the equilibrium-based assay. Using this assay, herein named the [³²P]ATP/PP_i assay, we followed the activation step of several AARSs. The obtained data are in good agreement with the previously published kinetic constants obtained with the standard ATP/[³²P]PP_i exchange assay.