Kinetics of Macrolide Action

Abstract

Members of the macrolide class of antibiotics inhibit peptide elongation on the ribosome by binding close to the peptidyltransferase center and blocking the peptide exit tunnel in the large ribosomal subunit. We have studied the modes of action of the macrolides josamycin, with a 16-membered lactone ring, and erythromycin, with a 14-membered lactone ring, in a cell-free mRNA translation system with pure components from Escherichia coli. We have found that the average lifetime on the ribosome is 3 h for josamycin and less than 2 min for erythromycin and that the dissociation constants for josamycin and erythromycin binding to the ribosome are 5.5 and 11 nM, respectively. Josamycin slows down formation of the first peptide bond of a nascent peptide in an amino acid-dependent way and completely inhibits formation of the second or third peptide bond, depending on peptide sequence. Erythromycin allows formation of longer peptide chains before the onset of inhibition. Both drugs stimulate the rate constants for drop-off of peptidyl-tRNA from the ribosome. In the josamycin case, drop-off is much faster than drug dissociation, whereas these rate constants are comparable in the erythromycin case. Therefore, at a saturating drug concentration, synthesis of fulllength proteins is completely shut down by josamycin but not by erythromycin. It is likely that the bacteriotoxic effects of the drugs are caused by a combination of inhibition of protein elongation, on the one hand, and depletion of the intracellular pools of aminoacyltRNAs available for protein synthesis by drop-off and incomplete peptidyl-tRNA hydrolase activity, on the other hand.

The Molecular Mechanism of Peptide-mediated Erythromycin Resistance

“…3A) and the rate constants obtained from our cell-free in vitro translation system (Table 2 and Ref. 21) are sufficient to fully account for the in vivo induced resistance in a large interval of erythromycin concentrations and peptide expression levels (Figs. 3, B and C (and inset)).…”

“…Expression of a mini-gene buried in the 23 S rRNA causes low level resistance to erythromycin (6), and it has been suggested that synthesis of this resistance peptide on an erythromycin-containing ribosome can clean it from the drug, thereby making an erythromycin-free 50 S subunit available for a new round of initiation of protein synthesis with another mRNA (8). When the nascent peptide is longer than six to eight amino acids, it covers the erythromycin binding site, which makes the ribosome refractory to further inhibition by erythromycin, allowing for synthesis of full-length proteins (3,21).…”

“…1). Because the two drugs have overlapping ribosomal binding sites (1,20,21), josamycin cannot bind and shut down peptide synthesis until after dissociation of erythromycin from the 50 S subunit. The josamycin concentration used in the chase (83 M) leads to an association rate of 2.7 s Ϫ1 (21).…”

“…We constructed a model for erythromycin resistance in bacterial populations (Fig. 3A) based on these and other parameters (listed in the supplemental material) for protein synthesis obtained from our cell-free mRNA translation system (21). The model (detailed description in the supplemental material) contains seven different states of the large ribosomal subunit (50 S) (Fig.…”

“…The value of the rate constant for dissociation of erythromycin from ribosomes synthesizing the control peptide was estimated as 0.01 s Ϫ1 (Fig. 1B), which corresponds to the rate constant for spontaneous dissociation of erythromycin from empty ribosomes (21). In contrast, the value of the rate constant for dissociation of erythromycin from ribosomes synthesizing the resistance peptide was estimated as 0.03 s Ϫ1 , a value coinciding with the rate (s Ϫ1 ) of pentapeptide synthesis per ribosome in the absence of josamycin (Fig.…”