Adenosine Tetraphosphoadenosine Drives a Continuous ATP-Release Assay for Aminoacyl-tRNA Synthetases and Other Adenylate-Forming Enzymes

Aminoacyl-tRNA synthetases are essential for the correct linkage of an amino acid to its cognate tRNA. The accuracy of this reaction is key to the fidelity of protein synthesis. Tractable, continuous assays are of value both in characterizing the functions of these enzymes and their exploitation as drug targets. Therefore, we have exploited the hitherto unexplored ability of these enzymes to consume the diadenosine nucleotide diadenosine 5’,5’’’ P1 P4 tetraphosphate (adenosine tetraphosphoadenosine (Ap4A)) with the concomitant production of ATP in a pyrophosphate and amino acid dependent manner to develop such an assay. We have used this assay to probe the stereo-selectivity of amino acid activation by isoleucyl-tRNA le and Valyl-tRNA Val synthetases and to identify analogues of intermediates in their catalytic pathway that might facilitate simultaneous targeting of multiple synthetases. Finally we report the utility of Ap4A based assays in the screening and subsequent identification of inhibitors of these enzymes, where the kinetics of Ap4A utilization allow the detection of inhibitors with nM to mM affinities.