Kinetic characterization of amino acid activation by aminoacyl‐tRNA synthetases using radiolabelled γ‐[32P]ATP
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 (PPi) formation. In a subsequent s...
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Wiley
2025-04-01
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| Online Access: | https://doi.org/10.1002/2211-5463.13903 |
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| author | Igor Živković Morana Dulic Petra Kozulic Marko Mocibob Ita Gruic‐Sovulj |
| author_facet | Igor Živković Morana Dulic Petra Kozulic Marko Mocibob Ita Gruic‐Sovulj |
| author_sort | Igor Živković |
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| description | 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 (PPi) 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]PPi exchange assay, which relies on the equilibrium exchange of radiolabel between PPi and ATP using [32P]PPi as a labelled compound. However, this method became much less convenient when [32P]PPi 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/PPi 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]PPi exchange assay. |
| format | Article |
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| institution | DOAJ |
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| language | English |
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| spelling | doaj-art-e1e1599bf44449c7b1a39b2e4a2adc6d2025-08-20T02:54:39ZengWileyFEBS Open Bio2211-54632025-04-0115458058610.1002/2211-5463.13903Kinetic characterization of amino acid activation by aminoacyl‐tRNA synthetases using radiolabelled γ‐[32P]ATPIgor Živković0Morana Dulic1Petra Kozulic2Marko Mocibob3Ita Gruic‐Sovulj4Department of Chemistry, Faculty of Science University of Zagreb Zagreb CroatiaDepartment of Chemistry, Faculty of Science University of Zagreb Zagreb CroatiaDepartment of Chemistry, Faculty of Science University of Zagreb Zagreb CroatiaDepartment of Chemistry, Faculty of Science University of Zagreb Zagreb CroatiaDepartment of Chemistry, Faculty of Science University of Zagreb Zagreb CroatiaAminoacyl‐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 (PPi) 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]PPi exchange assay, which relies on the equilibrium exchange of radiolabel between PPi and ATP using [32P]PPi as a labelled compound. However, this method became much less convenient when [32P]PPi 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/PPi 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]PPi exchange assay.https://doi.org/10.1002/2211-5463.13903amino acid activationaminoacyl‐tRNA synthetasesATP/PPi exchangeisotopic equilibrium exchange |
| spellingShingle | Igor Živković Morana Dulic Petra Kozulic Marko Mocibob Ita Gruic‐Sovulj Kinetic characterization of amino acid activation by aminoacyl‐tRNA synthetases using radiolabelled γ‐[32P]ATP FEBS Open Bio amino acid activation aminoacyl‐tRNA synthetases ATP/PPi exchange isotopic equilibrium exchange |
| title | Kinetic characterization of amino acid activation by aminoacyl‐tRNA synthetases using radiolabelled γ‐[32P]ATP |
| title_full | Kinetic characterization of amino acid activation by aminoacyl‐tRNA synthetases using radiolabelled γ‐[32P]ATP |
| title_fullStr | Kinetic characterization of amino acid activation by aminoacyl‐tRNA synthetases using radiolabelled γ‐[32P]ATP |
| title_full_unstemmed | Kinetic characterization of amino acid activation by aminoacyl‐tRNA synthetases using radiolabelled γ‐[32P]ATP |
| title_short | Kinetic characterization of amino acid activation by aminoacyl‐tRNA synthetases using radiolabelled γ‐[32P]ATP |
| title_sort | kinetic characterization of amino acid activation by aminoacyl trna synthetases using radiolabelled γ 32p atp |
| topic | amino acid activation aminoacyl‐tRNA synthetases ATP/PPi exchange isotopic equilibrium exchange |
| url | https://doi.org/10.1002/2211-5463.13903 |
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