Chemical characterization of ethambutol binding to Mycobacterium smegmatis

Recent studies showed that critical binding of ethambutol to Mycobacterium smegmatis was both inhibited and reversed by ions. This apparent ion-susceptible characteristic suggested that ethambutol is held at critical sites by electrostatic bonds. Dissociation constant, pH, and ionic strength studies were designed to further characterize ethambutol binding. M. smegmatis was grown in Sauton synthetic liquid medium (pH 7.4) under aerated conditions at 37 C. The pH or ionic strength of the medium was modified to meet the needs of particular experiments. Titration data revealed that ethambutol dihydrochloride has two apparent dissociation constants (pKa(1) = 6.35, pKa(2) = 9.35). Uptake experiments, in which pH was varied, showed that dihydrochloride and free base ethambutol were bound to a greater extent than the monohydrochloride. However, dihydrochloride and free base binding were not related to biological activity. Ethambutol exerted its maximal growth inhibitory effect at pH values near neutrality, where it exists primarily as the monohydrochloride and showed minimal binding. The increased ethambutol binding observed at pH 7.4 in media of lowered ionic strength was consistent with growth studies showing a reduction in the minimal inhibitory growth concentration in such media. However, nonspecific as well as critical binding was enhanced at low ionic strength. We conclude that binding of ethambutol by M. smegmatis involves a heterogeneous group of drug binding sites, only one of which is directly related to biological activity. Although nothing is known about the ethambutol target site itself, critical binding of the drug seems to require the single positively charged monohydrochloride form. Both hydrogen bonds and ionic linkages are probably involved.