Role of protonated and neutral forms of macrolides in binding to ribosomes from gram-positive and gram-negative bacteria.

Erythromycin binds to a single site on the bacterial 50S ribosomal subunit and perturbs protein synthesis. However, erythromycin contains desosamine and thus exists in both protonated (greater than 96%) and neutral (less than 4%) forms at physiological pH because of the pKa of the dimethylamino group. We therefore examined the relative roles of both forms in binding to ribosomes isolated from two species each of gram-positive and gram-negative bacteria. We developed a system to directly measure the forward (association) rate constant of formation of the macrolide-ribosome complex, and we have measured both the forward and reverse (dissociation) rate constants as a function of pH. Forward rate constants and binding affinity did not correlate with pH when the interaction of erythromycin with ribosomes from both gram-positive and gram-negative bacteria was examined, demonstrating that the protonated form of this macrolide binds to ribosomes. Conversely, the neutral form of macrolide cannot be the sole binding species and appears to bind with the same kinetics as the protonated form. Forward rate constants were 3- to 4-fold greater at physiological pH, and binding affinity calculated from rate constants was 5- to 10-fold greater than previously estimated. Similar results were obtained with azithromycin, a novel 15-membered macrolide that contains an additional tertiary amine in the macrolide ring. Ribosome- and macrolide-specific kinetic parameters were demonstrated at neutral pH and may be related to the potency of the two macrolides against gram-positive and gram-negative bacteria.