Dissociation kinetics of complexes between the antibiotic rifamycin and DNA-dependent RNA polymerase from Escherichia coli.
Phosphocellulose chromatography was employed to measure the binding of 3-(2-[14C]acetamidoethyl)-thiorifamycin(abbreviated[14C]AcNHEtS-Rif) to RNA polymerase core enzyme. The technique yielded a correct value for the stoichiometry of interaction. The same method was successfully applied to an investigation of the dissociation kinetics of the [14C]AcNHEtS-Rif - core-polymerase complex. We observed biphasic dissociation kinetics not in agreement with the existence of one single first-order dissociation step. Assuming two independent dissociation reactions, rate constants have been evaluated differing roughly by 10-fold (1.7 X 10(-3) s-1 and 1.5 X 10(-4) s-1 at 25 degrees C). The ratio of the amplitudes of the biphasic dissociation kinetics changed with temperature. The kinetic data are interpreted as indirect evidence for the existence of two enzyme species differing in their dynamic properties with respect to the binding of the antibiotic rifamycin. Our data furthermore lend support to the assumption that the two enzyme forms are in equilibrium. The observed sigmoidal dependence of the dissociation rate on temperature could indicate a conformational transition of RNA core polymerase with a transition midpoint at around 20 degrees C. The dye, rose bengal, was found to be as effective as AcNHEtS-Rif itself as a competing agent. The dissociation kinetics of the [14C]-AcNHEtS-Rif - core-polymerase complex in the presence of an excess of the dye rose bengal were found to be very similar to those measured in the presence of AcNHEtS-Rif. This could mean that rose bengal and AcNHEtS-Rif compete for the same site at RNA core polymerase. The dissociation of the ternary complex [14C]AcNHEtS-Rif - core-polymerase - poly[d(A-T)] was followed by gel filtration. Up to the extent of dissociation measured, the reaction appeared to follow first-order kinetics. The dissociation rate constant was calculated to be 1.7 X 10(-4) s-1. Experiments to determine the effect of the antibiotic streptolydigin on the dissociation kinetics of the [14C]AcNHEtS-Rif - core-polymerase complex have not led to conclusive results.
D001665:Binding Sites; D001667:Binding, Competitive; D055598:Chemical Phenomena; D002621:Chemistry; D002845:Chromatography; D012321:DNA-Directed RNA Polymerases; D004926:Escherichia coli / Q000201:enzymology; D066298:In Vitro Techniques; D007700:Kinetics; D008956:Models, Chemical; D011485:Protein Binding; D012294:Rifamycins; D012395:Rose Bengal; D013696:Temperature
D012294:Rifamycins; D012395:Rose Bengal; D012321:DNA-Directed RNA Polymerases