title: Adenosine tetraphosphoadenosine drives a continuous ATP-release assay for aminoacyl-tRNA synthetases and other adenylate-forming enzymes.
abstract: 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',5‴ P(1) P(4) tetraphosphate; Ap4A) 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 Ap4A-based assays for identification of synthetase inhibitors with nanomolar to millimolar affinities. Finally, we demonstrate the broad application of Ap4A utilization with a continuous Ap4A-driven RNA ligase assay.
authors.affiliation: School of Life Sciences, University of Warwick , Gibbet Hill Road, Coventry, West Midlands CV4 7AL, U.K.
authors.forename: Adrian J
authors.initials: AJ
authors.lastname: Lloyd
authors.forename: Nicola J
authors.initials: NJ
authors.lastname: Potter
authors.forename: Colin W G
authors.initials: CW
authors.lastname: Fishwick
authors.forename: David I
authors.initials: DI
authors.lastname: Roper
authors.forename: Christopher G
authors.initials: CG
authors.lastname: Dowson
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000604:Amino Acyl-tRNA Synthetases / Q000032:analysis* / Q000737:chemistry; D001681:Biological Assay; D015226:Dinucleoside Phosphates / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D013379:Substrate Specificity
chemical_list: D015226:Dinucleoside Phosphates; C020733:diadenosine tetraphosphate; D000255:Adenosine Triphosphate; D000604:Amino Acyl-tRNA Synthetases