Kinetic Studies of the Interaction between Rifampicin and DNA‐Dependent RNA Polymerase of Escherichia coli

Rifampicin specifically inhibits bacterial RNA polymerase by forming a tight complex with the enzyme in a stoichiometric ratio of 1:1. In the present work, dextran‐coated charcoal was used as a tool to quantify the kinetic rate constants of the association and dissociation of this complex. With this simple and sensitive assay, the rate of dissocaition (kd) of the enzyme · antibiotic complex was shown to follow first‐order kinetics, with a half‐life of 12 h at 0°C, which decreases to 9 min at 37°C. The rate of association follows second‐order kinetics, with a rate constant (ka) of 6 × 104 M−1 s−1 at 0°C, which increases to 1.2 × 106 M−1 s−1 at 37°C. From the kd/ka ratio, the equilibrium constant K has been calculated to be 3.5 × 10−10 M at 0°C and 10−9 M at 37°C. The physical nature of the bonds involved in the rifampicin · RNA‐polymerase complex seems to be mainly lipophilic, since high ionic strength had no influence on ka and even decreased kd, whereas dimethylsulfoxide (Me2SO), a less polar solvent than water, decreased ka and increased kd, resulting in an up to 20 times higher K. The close correlation between binding of rifampicin to RNA polymerase and enzyme inhibition by the drug can be demonstrated directly: at ascending concentrations of Me2SO there is a good correlation between the rise in K and the increase in the amount of rifampicin required for 50% enzyme inhibition.