Erythromycin, roxithromycin, and clarithromycin: use of slow-binding kinetics to compare their in vitro interaction with a bacterial ribosomal complex active in peptide bond formation.
In a cell-free system derived from Escherichia coli, it is shown that clarithromycin and roxithromycin, like their parent compound erythromycin, do not inhibit the puromycin reaction (i.e., the peptide bond formation between puromycin and AcPhe-tRNA bound at the P-site of 70S ribosomes programmed with heteropolymeric mRNA). Nevertheless, all three antibiotics compete for binding on the ribosome with tylosin, a 16-membered ring macrolide that behaves as a slow-binding, slowly reversible inhibitor of peptidyltransferase. The mutually exclusive binding of these macrolides to ribosomes is also corroborated by the fact that they protect overlapping sites in domain V of 23S rRNA from chemical modification by dimethyl sulfate. From this competition effect, detailed kinetic analysis revealed that roxithromycin or clarithromycin (A), like erythromycin, reacts rapidly with AcPhe-tRNA.MF-mRNA x 70S ribosomal complex (C) to form the encounter complex CA which is then slowly isomerized to a more tight complex, termed C*A. The value of the overall dissociation constant, K, encompassing both steps of macrolide interaction with complex C, is 36 nM for erythromycin, 20 nM for roxithromycin, and 8 nM for clarithromycin. Because the off-rate constant of C*A complex does not significantly differ among the three macrolides, the superiority of clarithromycin as an inhibitor of translation in E. coli cells and many Gram-positive bacteria may be correlated with its greater rate of association with ribosomes.
Dinos
George P
GP
Laboratory of Biochemistry, School of Medicine, University of Patras, Patras, Greece. geodinos@med.upatras.gr
Connell
Sean R
SR
Nierhaus
Knud H
KH
Kalpaxis
Dimitrios L
DL
D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D001419:Bacteria / Q000187:drug effects* / Q000378:metabolism; D017291:Clarithromycin / Q000737:chemistry / Q000494:pharmacology; D004917:Erythromycin / Q000737:chemistry / Q000494:pharmacology; D007700:Kinetics; D012270:Ribosomes / Q000187:drug effects* / Q000378:metabolism; D015575:Roxithromycin / Q000737:chemistry / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D015575:Roxithromycin; D004917:Erythromycin; D017291:Clarithromycin