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

Biochemistry. 2000 Sep 26;39(38):11621-8. doi: 10.1021/bi000811f.

Abstract

The inhibition of peptide bond formation by tylosin, a 16-membered ring macrolide, was studied in a model system derived from Escherichia coli. In this cell-free system, a peptide bond is formed between puromycin (acceptor substrate) and AcPhe-tRNA (donor substrate) bound at the P-site of poly(U)-programmed ribosomes. It is shown that tylosin inhibits puromycin reaction as a slow-binding, slowly reversible inhibitor. Detailed kinetic analysis reveals that tylosin (I) reacts rapidly with complex C, i.e., the AcPhe-tRNA. poly(U).70S ribosome complex, to form the encounter complex CI, which then undergoes a slow isomerization and is converted to a tight complex, CI, inactive toward puromycin. These events are described by the scheme C + I <==> (K(i)) CI <==> (k(4), k(5)) CI. The K(i), k(4), and k(5) values are equal to 3 microM, 1.5 min(-1), and 2.5 x 10(-3) min(-1), respectively. The extremely low value of k(5) implies that the inactivation of complex C by tylosin is almost irreversible. The irreversibility of the tylosin effect on peptide bond formation is significant for the interpretation of this antibiotic's therapeutic properties; it also renders the tylosin reaction a useful tool in the study of other macrolides failing to inhibit the puromycin reaction but competing with tylosin for common binding sites on the ribosome. Thus, the tylosin reaction, in conjunction with the puromycin reaction, was applied to investigate the erythromycin mode of action. It is shown that erythromycin (Er), like tylosin, interacts with complex C according to the kinetic scheme C + Er <==> (K(er)) CEr <==> (k(6), k(7)) C*Er and forms a tight complex, CEr, which remains active toward puromycin. The determination of K(er), k(6), and k(7) enables us to classify erythromycin as a slow-binding ligand of ribosomes.

MeSH terms

  • Anti-Bacterial Agents / chemistry
  • Anti-Bacterial Agents / metabolism*
  • Binding, Competitive
  • Drug Synergism
  • Erythromycin / chemistry
  • Erythromycin / metabolism*
  • Kinetics
  • Peptide Biosynthesis / drug effects
  • Peptides / antagonists & inhibitors
  • Peptides / chemistry
  • Peptides / metabolism*
  • Protein Synthesis Inhibitors / chemistry
  • Puromycin / chemistry
  • Ribosomal Proteins / antagonists & inhibitors
  • Ribosomal Proteins / chemistry
  • Ribosomal Proteins / metabolism*
  • Ribosomes / chemistry
  • Ribosomes / drug effects
  • Ribosomes / metabolism*
  • Solutions
  • Tylosin / chemistry
  • Tylosin / metabolism*

Substances

  • Anti-Bacterial Agents
  • Peptides
  • Protein Synthesis Inhibitors
  • Ribosomal Proteins
  • Solutions
  • Puromycin
  • Erythromycin
  • Tylosin