Adenosine 5′-O-(3-thio)triphosphate (ATPγS) is a substrate for the nucleotide hydrolysis and RNA unwinding activities of eukaryotic translation initiation factor eIF4A

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Abstract
Whereas ATPγS is often considered a nonhydrolyzable substrate for ATPases, we present evidence that ATPγS is a good substrate for the RNA-stimulated nucleotide hydrolysis and RNA unwinding activities of eIF4A. In the presence of saturating single-stranded poly(U) RNA, eIF4A hydrolyzes ATPγS•Mg and ATP•Mg with similar steady-state parameters (KMNTP•Mg = 66 and 58 μM and kcat = 1.0 and 0.97 min−1, respectively). ATPγS•Mg also supports catalysis of RNA unwinding within 10-fold of the rate supported by ATP•Mg. The identical steady-state rate parameters, in comparison with the expected difference in the intrinsic rate of hydrolysis for ATP and ATPγS, suggest a nonchemical rate-limiting step for nucleotide hydrolysis. These results raise caution concerning the assumption that ATPγS is a nonhydrolyzable ATP analog and underscore the utility of thio-substituted NTPs as mechanistic probes.