Synthesis of guanosine 5'-di- and -triphosphate derivatives with modified terminal phosphates. Effect on ribosome-elongation factor G-dependent reactions

Abstract

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.

Synthesis, characterization and application of two nucleoside triphosphate analogues, GTPγNH2 and GTPγF

“…the c-(4-azido) anilide of GTP to substitute efficiently for GTP as a photoaffinity label in the elongation factor protein EF-Tu [32]. The GTPcF substrate analogue was first prepared by Eckstein et al [9], by the method of Haley & Yount [33], and used to study its interaction with the GTP-binding site of adenylyl cyclase [34]. We synthesized this substance by the simple method of Wittmann [10], which works very well with adenine nucleotides.…”

Mechanism of the Inhibition of Protein Synthesis by Kirromycin. Role of Elongation Factor Tu and Ribosomes

“…Biological components, materials and methods used which are not described in this section or in the legends, are the same as reported in the preceding 3H-labeled elongation factors were prepared by growing Escherichiu coli BT2' in a minimal medium containing [3H]lysine or ['Hlvaline [4]. The cell paste was disrupted in a small mortar with a pestle by hand, using otherwise the described procedure [5].…”

“…Poly(U)-directed poly(phenyla1anine) synthesis and ribosome-dependent EF-G GTPase activity were measured as described [2,4]. Mocimycin fragments 2b (an apolar diene) and 4 a (a polar compound), obtained by splitting the antibiotic molecule with acetic acid treatment at room temperature for several days [9], were provided by Dr R. Beukers (Gist-Brocades N.V., Research and Development, Delft, The Netherlands).…”

Topology of the GTP‐binding site of adenylyl cyclase from pigeon erythrocytes

“…*The splitting constant JP-F of GDP(13F) and GTP(~,F) is not 450 Hz as erroneously reported [7] but 900 Hz.…”