Abstract
The kinetics of the interaction between the 50 S subunits (R) of bacterial ribosomes and the antibiotics virginiamycin S (VS), virginiamycin M (VM), and erythromycin have been studied by stopped flow fluorimetric analysis, based on the enhancement of VS fluorescence upon its binding to the 50 S ribosomal subunit. Virginiamycin components M and S exhibit a synergistic effect in vivo, which is characterized in vitro by a 5- to 10-fold increase of the affinity of ribosomes for VS, and by the loss of the ability of erythromycin to displace VS subsequent to the conformational change (from R to R*) produced by transient contact of ribosomes with VM. Our kinetic studies show that the VM-induced increase of the ribosomal affinity for VS (K*VS = 25 X 10(6) M-1 instead of KVS = 5.5 X 10(6) M-1) is due to a decrease of the dissociation rate constant (k*-VS = 0.008 s-1 instead of 0.04 s-1). The association rate constant remains practically the same (k+VS approximately k*+VS = 2.8 X 10(5) M-1 s-1), irrespective of the presence of VM. VS and erythromycin bind competitively to ribosomes. This effect has been exploited to determine the dissociation rate constant of VS directly by displacement experiments from VS . 50 S complexes, and the association rate constant of erythromycin (k+Ery = 3.2 X 10(5) M-1 S-1) on the basis of competition experiments for binding of free erythromycin and VS to ribosomes. By making use of the change in competition behavior of erythromycin and VS, after interaction of ribosomes with VM, the conformational change induced by VM has been explored. Within the experimentally available concentration region, the catalytic effect of VM has been shown to be coupled to its binding kinetics, and the association rate constant of VM has been determined (k+VM = 1.4 X 10(4) M-1 S-1). Evidence is presented for a low affinity binding of erythromycin (K*Ery approximately 3.3 X 10(4) M-1) to ribosomes altered by contact with VM. A model involving a sequence of 5 reactions has been proposed to explain the replacement of ribosome-bound erythromycin by VS upon contact of 50 S subunits with VM.
Highlights
Thekinetics of the interaction between the 50 S sensitive bacteria
Binding of virginiamycin S (VS) to 50 S Ribosomal Subunits-We have previously shown [18]that VS binding to 50 S ribosomal subunits produces an increase of the fluorescence emission of VS.Kinetics of binding was established by fluorescence stopped flow measurements of mixtures with constant ribosome concentration (0.5 p ~ an)d increasing VS concentrations (5 to20 p ~ ) .the experiments were always done in pseudo first order conditions
Kinetics of virginiamycin M (VM)-induced Ribosomes Alteration-We have previously reported that Erythromycin is unable to remove VS from ribosomes pretreatedwith virginiamycinM [23]
Summary
Ribosomes were withdrawn and centrifuged for 4 hat 300,000 X g (angular rotor 50.2 Ti from Spinco). Pellets of subunits, collected by centrifugation, were suspended in buffer A 1 and centrifuged at 10,000 X g for 20 min a t 4 "C, and supernatantswere stored inliquid nitrogen [14]. Equilibrium Binding Assay for VirginiamycinS-Complexes of virginiamycin S with 50 S subunits were measured by the increase of fluorescence intensity (AF41s.m).The latter was previously shown to be proportional to theconcentration of the particle-bound antibiotic [18]. For this purpose, 50 S subunits in buffer A1 were reactivated (20-min incubation a t 45 "C) and incubated in buffer B with increasing concentrations of virginiamycin S. All kinetics measurements were performed in buffer B at 25 "C
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