Kinetic analysis of the interaction of guanine nucleotides with eukaryotic translation initiation factor eIF5B

Eukaryotic translation initiation factor eIF5B is a ribosome-dependent GTPase that is responsible for the final step in initiation, which involves the displacement of initiation factors from the 40S ribosomal subunit in initiation complexes and its joining with the 60S subunit. Hydrolysis of eIF5B-bound GTP is not required for its function in subunit joining but is necessary for the subsequent release of eIF5B from assembled 80S ribosomes. Here we investigated the kinetics of guanine nucleotide binding to eIF5B by a fluorescent stopped-flow technique using fluorescent mant derivatives of GTP and GDP and of the GTP analogues GTPγS and GMPPNP. The affinity of eIF5B for mant-GTP (K_d ∼ 14-18 μM) was approximately 7-fold less than for mant-GDP (K_d ∼ 2.3 μM), and both guanine nucleotides dissociated rapidly from eIF5B (k_-1^mant-GTP ∼ 22-28 s^-1, k_-1^mant-GDP ∼ 10-14 s^-1). These properties of eIF5B suggest a rapid spontaneous GTP/GDP exchange on eIF5B and are therefore consistent with it having no requirement for a special guanine nucleotide exchange factor. The affinity of eIF5B for mant-GTPγS was about 2 times lower (K_d ∼ 6.9 μM) and for mant-GMPPNP 1.5 times higher (K_d ∼ 25.7 μM) than for mant-GTP, indicating that eIF5B tolerates modifications of the triphosphate moiety well.