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

作者：Lloyd Adrian J^*; Potter Nicola J; Fishwick Colin W G; Roper David I; Dowson Christopher G

来源：ACS Chemical Biology, 2013, 8(10): 2157-2163.

DOI：10.1021/cb400248f

Aminoacyl-tRNA synthetases are essential for the correct linkage of amino acids to cognate tRNAs to maintain the fidelity of protein synthesis. Tractable, continuous assays are valuable for characterizing the functions of synthetases and for their exploitation as drug targets. We have exploited the unexplored ability of these enzymes to consume adenosine tetraphosphoadenosine (diadenosine 5%26apos;,5 %26apos;%26apos; P-1 P-4 tetraphosphate; Ap(4)A) and produce ATP to develop such an assay. We have used this assay to probe the stereoselectivity of isoleucyl-tRNA(Ile) and Valyl-tRNA(Val) synthetases and the impact of tRNA on editing by isoleucyl-tRNA(Ile) synthetase (IleRS) and to identify analogues of intermediates of these enzymes that might allow targeting of multiple synthetases. We further report the utility of Ap(4)A-based assays for identification of synthetase inhibitors with nanomolar to millimolar affinities. Finally, we demonstrate the broad application of Ap(4)A utilization with a continuous Ap(4)A-driven RNA ligase assay.

• 出版日期2013-10