Affinity labelling of the eukaryotic elongation factor EF-2 with the guanosine nucleotide analogue 5'-p-fluorosulfonylbenzoylguanosine.
During the translocation of the nascent peptide chain from the ribosomal aminoacyl-site to the peptidyl-site, GTP is hydrolyzed by a mechanism dependent on both ribosomes and the elongation factor EF-2. For insight into the mechanism of GTP hydrolysis, we studied the ability of the GTP analogue 5'-p- fluorosulfonylbenzoylguanosine ( FSO2BzGuo ) to act as an affinity label of the guanine-specific site. Pre-incubation of EF-2 with FSO2BzGuo at increasing concentrations progressively inactivated the EF-2 and ribosome-dependent GTPase activity. Up to 0.5 mM FSO2BzGuo , the inactivation of the GTPase activity was stoichiometrically correlated with the covalent binding of [3H] FSO2BzGuo . Thus, one molecule of covalently bound FSO2BzGuo completely inactivated the GTPase activity of EF-2. Ribosomes or 60-S ribosomal subunits pre-incubated with FSO2BzGuo were not inactivated, consistent with the idea that the GTP hydrolysis involved in the ribosomal translocation takes place on EF-2.
D000345:Affinity Labels; D000818:Animals; D001667:Binding, Competitive; D006151:Guanosine / Q000031:analogs & derivatives* / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008862:Microsomes, Liver / Q000032:analysis; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D012270:Ribosomes / Q000378:metabolism
C017320:5'-(4-fluorosulfonylbenzoyl)guanosine; D000345:Affinity Labels; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D006151:Guanosine; D006160:Guanosine Triphosphate; D008274:Magnesium