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

Abstract

Whereas ATPgammaS is often considered a nonhydrolyzable substrate for ATPases, we present evidence that ATPgammaS 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 ATPgammaS.Mg and ATP.Mg with similar steady-state parameters (KM(NTP.Mg) = 66 and 58 microM and kcat = 1.0 and 0.97 min(-1), respectively). ATPgammaS.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 ATPgammaS, suggest a nonchemical rate-limiting step for nucleotide hydrolysis. These results raise caution concerning the assumption that ATPgammaS is a nonhydrolyzable ATP analog and underscore the utility of thio-substituted NTPs as mechanistic probes.

Characterization of a heat-stable enzyme possessing GTP-dependent RNA ligase activity from a hyperthermophilic archaeon, Pyrococcus furiosus

“…2D). Since it is reported that g-S NTPs are capable of serving as a substrate for nucleotide hydrolysis (Shuman et al 1980;Peck and Herschlag 2003), we further conducted a trace experiment using fluorescence-labeled GTP (Mant-GTP) and confirmed that GTP hydrolysis did not occur during the ligation step (although we detected almost the same ligation activity as GTP). The observation was also confirmed in our NMR experiment (data not shown).…”

The mechanism of ATP-dependent RNA unwinding by DEAD box proteins

“…Dissection of individual steps in the nucleotide cycle of DbpA has provided a kinetic and thermodynamic framework for the nucleotide cycle and its links to RNA binding and unwinding (Henn et al, 2008). Phosphate release was identified as a rate-limiting step both in the absence and in the presence of RNA (Henn et al, 2008), in line with similar rates of ATP-and ATPgS-supported RNA unwinding (Peck and Herschlag, 2003). Recently, the effect of the ATP analogs ADP-BeF x and ADP-AlF x on RNA unwinding has been examined (Liu et al, 2008).…”

“…ADPNP is not hydrolyzed by DEAD box proteins and does not support RNA unwinding (Liu et al, 2008;Theissen et al, 2008). In contrast, ATPgS, which is frequently used as a 'nonhydrolyzable' nucleotide analog for many ATPases, is efficiently hydrolyzed by eIF4A and supports RNA unwinding (Peck and Herschlag, 2003). Although ATPgS is hydrolyzed 10-fold more slowly than ATP, the unwinding rates with ATP-and ATPgS are similar, suggesting that the chemical step of ATP hydrolysis is not rate limiting for RNA unwinding (Peck and Herschlag, 2003).…”

“…In contrast, ATPgS, which is frequently used as a 'nonhydrolyzable' nucleotide analog for many ATPases, is efficiently hydrolyzed by eIF4A and supports RNA unwinding (Peck and Herschlag, 2003). Although ATPgS is hydrolyzed 10-fold more slowly than ATP, the unwinding rates with ATP-and ATPgS are similar, suggesting that the chemical step of ATP hydrolysis is not rate limiting for RNA unwinding (Peck and Herschlag, 2003). Dissection of individual steps in the nucleotide cycle of DbpA has provided a kinetic and thermodynamic framework for the nucleotide cycle and its links to RNA binding and unwinding (Henn et al, 2008).…”

Interactions between eIF4AI and its accessory factors eIF4B and eIF4H

“…32 P-59-end-labeled 12-nt RNA and unlabeled 32-nt RNA were mixed in a 1.75:1 molar ratio, and hybridized duplexes prepared as previously described (Peck and Herschlag 2003). Helicase assays were performed in Buffer B with 0.1-0.2 nM duplex, 1 mM Mg 2+ dATP, 0.5 mg/mL BSA, and 1 mM of the indicated proteins.…”