ENACYLOXIN IIA, AN INHIBITOR OF PROTEIN-BIOSYNTHESIS THAT ACTS ON ELONGATION-FACTOR TU AND THE RIBOSOME

This work analyzes the action of enacyloxin IIa, an inhibitor of bacte rial protein biosynthesis, Enacyloxin IIa [IC50 on poly(Phe) synthesis similar to 70 nM] is shown to affect the interaction between elongati on factor (EF) Tu and GTP or GDP; in particular, the dissociation of E F-Tu GTP is strongly retarded, causing the K-d of EF-Tu . GTP to decre ase from 500 to 0.7 nM, In its presence, the migration velocity of bot h GTP- and GDP-bound EF-Tu on native PAGE is increased, The stimulatio n of EF-Tu . GDP dissociation by EF-Ts is inhibited, EF-Tu . GTP can s till form a stable complex with aminoacyl-tRNA (aa-tRNA), but it no lo nger protects aa-tRNA against spontaneous deacylation, showing that th e EF-Tu . GTP orientation with respect to the 3' end of aa-tRNA is mod ified, However, the EF-Tu-dependent binding of aa-tRNA to the ribosoma l A-site is impaired only slightly by the antibiotic and the activity of the peptidyl-transferase center, as determined by puromycin reactiv ity, is not affected, In contrast, the C-terminal incorporation of Phe into poly(Phe)-tRNA bound to the P-site is inhibited, an effect that is observed if Phe-tRNA is bound to the A-site non-enzymatically as we ll, Thus, enacyloxin IIa can affect both EF-Tu and the ribosomal A-sit e directly, inducing an anomalous positioning of aa-tRNA, that inhibit s the incorporation of the amino acid into the polypeptide chain, Ther efore, it is the first antibiotic found to have a dual specificity tar geted to EF-Tu and the ribosome.