Synthesis of guanosine 5'-di- and -triphosphate derivatives with modified terminal phosphates: effect on ribosome-elongation factor G-dependent reactions.
A series of GTP and GDP analogues modified in the terminal phosphate has been synthesized and their activities were investigated in elongation factor G dependent reactions. All of the analogues, with the exception of guanosine 5'-O-(3-thiotriphosphate), were not hydrolyzed by EF-G and ribosomes, but were competitive inhibitors of the ribosome-dependent EF-G GTPase. The most active inhibitors were P3-fluoro P1-5'-guanosine triphosphate and P3-methyl P1-5'-guanosine triphosphate with a Ki of 1.0 X 10(-6) and 2.5 X 10(-6) M, respectively. The activity of the GTP alkyl ester derivatives decreased with increasing number of carbon atoms in the side chain. GTP analogues were much more effective inhibitors than the corresponding GDP derivatives. This points out the necessity of the presence of at least three negative charges in the phosphate chain of the nucleotide for an effective interaction with the active site of the ribosomal EF-G GTPase. Guanosine 5'-O-(3-thiotriphosphate), which was hydrolyzed at one-third the rate of GTP, was able to support poly(U)-directed poly(phenylalanine) polymerization. Possible mechanisms of ribosome-EF-G GTP hydrolysis that arise from our results are discussed. Activity of the nucleotide analogues in EF-G-ribosome complex formation compared well with their ability to inhibit ribosome-dependent EF-G GTPase, P3-fluoro P1-5'-guanosine triphosphate and P3-methyl P1-5'-guanosine triphosphate being again the most effective ones. The stabilizing action of fusidic acid on the EF-G-ribosome complex formation induced by the various nucleotides could not be correlated to any of the structural modifications of the substrate. Guanylyl methylene diphosphonate was displaced more readily than GDP from the EF-G-ribosome complex by GTP analogues insensitive to fusidic acid.
D004926:Escherichia coli / Q000378:metabolism; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000378:metabolism; D006150:Guanine Nucleotides / Q000138:chemical synthesis* / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D007700:Kinetics; D010441:Peptide Chain Elongation, Translational / Q000187:drug effects; D010445:Peptide Elongation Factors; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism*; D013329:Structure-Activity Relationship
D006150:Guanine Nucleotides; D010445:Peptide Elongation Factors; D006160:Guanosine Triphosphate; D020963:GTP Phosphohydrolase-Linked Elongation Factors