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

eIF4A catalyzes RNA-dependent ATPγS•Mg hydrolysis. (A) Fraction ATPγS•Mg hydrolyzed versus time in reactions containing 1 μM ATPγS•Mg, 0.25 μM eIF4A, and either no added RNA (○) or 25 μM poly(U) RNA (□). The slopes of linear fits yield observed rate constants (k_obs,hyd) of 2.6 × 10⁻⁵ and 4.8 × 10⁻³ min⁻¹, respectively. (B) Dependence of hydrolysis activity (k_cat,app) on the concentration of ATPγS•Mg (•) or ATP•Mg (○) in the presence of saturating poly(U) RNA (200 μM) and 0.25 μM eIF4A. The curves represent fits to the Michaelis-Menten model, yielding K_M^ATPγS•Mg = 66 ± 9 μM, K_M^ATP•Mg = 58 ± 11 μM, k_cat^ATPγS•Mg = 1.0 ± 0.1 and k_cat^ATP•Mg = 0.97 ± 0.06 min⁻¹.