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 GTPgammaS and GMPPNP. The affinity of eIF5B for mant-GTP (Kd approximately 14-18 microM) was approximately 7-fold less than for mant-GDP (Kd approximately 2.3 microM), and both guanine nucleotides dissociated rapidly from eIF5B (k-1mant-GTP approximately 22-28 s-1, k-1mant-GDP approximately 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-GTPgammaS was about 2 times lower (Kd approximately 6.9 microM) and for mant-GMPPNP 1.5 times higher (Kd approximately 25.7 microM) than for mant-GTP, indicating that eIF5B tolerates modifications of the triphosphate moiety well.
Pisareva
Vera P
VP
Department of Microbiology and Immunology, State University of New York Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, New York 11203, USA.
Hellen
Christopher U T
CU
Pestova
Tatyana V
TV
D000818:Animals; D039642:Eukaryotic Initiation Factors / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D011817:Rabbits
D039642:Eukaryotic Initiation Factors; D006150:Guanine Nucleotides; C451459:eukaryotic initiation factor-5B; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate