Adenosine tetraphosphoadenosine drives a continuous ATP-release assay for aminoacyl-tRNA synthetases and other adenylate-forming enzymes.
Adenosine tetraphosphoadenosine drives a continuous ATP-release assay for aminoacyl-tRNA synthetases and other adenylate-forming enzymes.
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DOI：
10.1021/cb400248f
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发表时间：
2013-10
影响因子：
4
通讯作者：
A. Lloyd;Nicola J Potter;C. Fishwick;D. Roper;C. Dowson
中科院分区：
文献类型：
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作者：
A. Lloyd;Nicola J Potter;C. Fishwick;D. Roper;C. Dowson
氨基酰基- trna合成酶是氨基酸正确连接同源trna以维持蛋白质合成保真度所必需的。易于处理的、连续的测定方法对于表征合成酶的功能及其作为药物靶点的开发是有价值的。我们已经利用了这些酶未开发的能力来消耗腺苷四磷酸腺苷(二腺苷5',5′，5′P(1) P(4)四磷酸；Ap4A)和产生ATP来发展这样的实验。我们使用该实验来探测异亮基tRNA（Ile）和缬氨酸tRNA（Val）合成酶的立体选择性，以及tRNA对异亮基tRNA（Ile）合成酶（IleRS）编辑的影响，并鉴定这些酶的中间产物的类似物，这些酶可能允许靶向多种合成酶。我们进一步报道了基于ap4的测定方法用于鉴定具有纳摩尔到毫摩尔亲和力的合成酶抑制剂的效用。最后，我们通过连续的Ap4A驱动RNA连接酶实验证明了Ap4A利用的广泛应用。
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.