Abstract 1804 Extending the scope of a coupled assay to measure alanyl-tRNA synthetase activity

Aminoacyl-tRNA synthetases are enzymes that catalyze the attachment of an amino acid to its corresponding tRNA, an essential step of protein synthesis. The most commonly used assay for monitoring aminoacylation catalyzed by alanyl-tRNA synthetase (AlaRS) utilizes radioactive alanine which allows for direct measurement of product formation, but is costly, time consuming, and requires sub-saturating concentrations of amino acid. We are interested in extending an assay developed for tyrosyl-tRNA synthetase1 that eliminates the need for radioactive amino acid by coupling the formation of AMP from the aminoacylation reaction to the reduction of NAD+ using two additional enzymes, AMP deaminase and IMP dehydrogenase. This would allow us to monitor the reaction by measuring NADH production with UV-Vis spectroscopy. To amplify the spectroscopic signal, we are attempting to regenerate the tRNA and amino acid substrates to keep the assay running continuously. Several substrate recycling strategies are available, including the use of stand-alone trans-editing enzymes2, which have previously been shown to deacylate aminoacylated tRNA products. 1. Richardson CJ, First EA. A continuous tyrosyl-tRNA synthetase assay that regenerates the tRNA substrate. Anal Biochem. 2015;486: 86 95. https://doi.org/10.1016/j.ab.2015.05.008 2. Nagy ABK, Bakhtina M, Musier-Forsyth K. Trans-editing by aminoacyl-tRNA synthetase-like editing domains. Enzym. 2020;48: 69–115. https://doi.org/10.1016/bs.enz.2020.07.002 Carleton College