Kinetic studies on the interaction between a ribosomal complex active in peptide bond formation and the macrolide antibiotics tylosin and erythromycin

The inhibition of peptide bond formation by tylosin, a 16-membered ring mac rolide, was studied in a model system derived from Escherichia coli. In thi s cell-free system, a peptide bond is formed between puromycin (acceptor su bstrate) and AcPhe-tRNA (donor substrate) bound at the P-site of poly(U)pro grammed ribosomes. It is shown that tylosin inhibits puromycin reaction as a slow-binding, slowly reversible inhibitor. Detailed kinetic analysis reve als that tylosin (I) reacts rapidly with complex C, i.e., the AcPhe-tRNA.po ly(U).7OS ribosome complex, to form the encounter complex CI, which then un dergoes a slow isomerization and is converted to a eight complex, C*I, inac tive toward puromycin. These events are described by the scheme C + I rever sible arrow (K-i) CI reversible arrow (k(4), k(5)) C*I. The K-i, k(4), and k(5) values are equal to 3 mu M, 1.5 min(-1), and 2.5 x 10(-3) min(-1), res pectively. The extremely low value of ks implies that the inactivation of c omplex C by tylosin is almost irreversible. The irreversibility of the tylo sin effect on peptide bond formation is significant fur the interpretation of this antibiotic's therapeutic properties; it also renders the tylosin re action a useful tool in the study of other macrolides failing to inhibit th e puromycin reaction but competing with tylosin for common binding sites on the ribosome. Thus, the tylosin reaction, in conjunction with the puromyci n reaction, was applied to investigate the erythromycin mode of action. It is shown that erythromycin (Er), like tylosin, interacts with complex C acc ording to the kinetic scheme C + Er reversible arrow (K-er) CEr reversible arrow (k(6), k(7)) C*Er and forms a tight complex, C*Er, which remains acti ve toward puromycin. The determination of K-er, k(6), and k(7) enables us t o classify erythromycin as a slow-binding ligand of ribosomes.