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

Whereas ATP S is often considered a nonhydrolyzable substrate for ATPases, we present evidence that ATP S 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 ATP S·Mg and ATP·Mg with similar steady-state parameters (KM NTP·Mg = 66 and 58 μM and kcat = 1.0 and 0.97 min , respectively). ATP S·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 ATP S, suggest a nonchemical rate-limiting step for nucleotide hydrolysis. These results raise caution concerning the assumption that ATP S is a nonhydrolyzable ATP analog and underscore the utility of thiosubstituted NTPs as mechanistic probes.