Binding of Dihydrostreptomycin to Escherichia coli Ribosomes: Kinetics of the Reaction

Abstract

Investigations were carried out on the binding of dihydrostreptomycin to purified (and reassociated) 70 S ribosomes and 30 S subunits from streptomycin-susceptible strains, and the results were compared with those of similar studies with native (run-off) 70 S ribosomes. At 0 C, only a small fraction of purified 70 S ribosomes and 30 S sub-units bound 1 molecule of the antibiotic tightly, and at a rate comparable to the binding occurring with native 70 S ribosomes. At temperatures of 10 C and above, there was a temperature-dependent increase in the extent of antibiotic binding to purified 70 S and 30 S particles up to a maximum of 1 molecule/ribosomal particle, but the kinetics of binding was slow in comparison to that taking place at 0 C. These and other results suggest that a major fraction of 30 S subunits and purified (or reassociated) 70 S ribosomes are inactive in binding the antibiotic. This has been localized to an instability of the free 30 S subunit, which in solution at 0 C has a half-life of 5 hr or less. Inactive 30 S or 70 S particles could be thermally activated, with the latter being identical in their streptomycin-binding properties to native 70 S ribosomes. The activation kinetics were slow in comparison to the binding kinetics for the antibiotic and were indicative of a conformational change in ribosomal structure. There thus appears to be a reversible transition between active and inactive forms of the ribosomal particles for streptomycin binding, but additional binding sites for the antibiotic are not created by the transitions. The active form of the 30 S subunit can be stabilized in the presence of polyuridylic acid, but much more effectively by association with the 50 S subunit to form a 70 S ribosome. The kinetics of dihydrostreptomycin binding were studied in both directions of the reaction, and the reaction in the direction of binding was found to be several orders of magnitude faster than that of the reverse, or debinding, direction. The kinetics of the exchange of bound dihydrostreptomycin with the free antibiotic were also determined and shown to have rate constants that are very similar to those of the debinding reaction, which is the rate-limiting step. It appears likely that the exchange reaction is proceeding via the same reaction pathway. The temperature dependence of the kinetics of dissociation of the bound complex was much greater than that in the direction of binding and accounted for most of the temperature dependence of the binding equilibrium. From the determined thermodynamic and activation parameters, it appears likely that binding of the antibiotic induces a conformational change in ribosomal structure to one that is less ordered than the native particle. Heterogeneity has been found in the kinetics of binding and of exchange, with a fraction of the 70 S population showing slower kinetics for both directions of the reaction.