Molecular Mechanism of the Rifampicin-RNA Polymerase Interaction

Lynwood R. Yarbrough, Felicia Y.H. Wu, Cheng Wen Wu

研究成果: Article同行評審

36 引文 斯高帕斯(Scopus)


Equilibrium and kinetic studies of the interaction of rifampicin with RNA polymerase of Escherichia coli were performed by exploiting the quenching of intrinsic fluorescence of the protein by the drug. Fluorimetric titrations show that rifampicin binds stoichiometrically to the core and holoenzyme with an apparent Kd of ≤ 3 × 10-9 M. Neither the addition of template nor the formation of the initiation complex in the presence of dinucleotide and nucleoside triphosphate prevents the rifampicin-enzyme interaction. Although the equilibrium binding constant for the rifampicin-RNA polymerase complex is about the same for the core and holoenzyme and the holoenzyme- T7 DNA complex, stopped-flow studies indicate that the rates at which rifampicin interacts with these enzyme forms are different. In all three cases, the kinetic data can be interpreted in terms of a mechanism in which the rapid bimolecular binding of rifampicin to RNA polymerase is followed by a relatively slow isomerization of the drug-enzyme complex:[formula omitted]While the values of dissociation constant, K1 = (K-1/K1), for the first binary complex (ER) are similar, the rate constant for the forward isomerization, k2, decreases in the order of core enzyme > holoenzyme > the holoenzyme-T7 DNA complex. The fact that this order is parallel to the relative rates of inactivation of the enzymes and the enzyme-DNA complex suggests that the inactivation may be due to the rifampicininduced isomerization (conformational change) of the enzyme. This is supported by our observation that an enzyme complex which is in the process of elongating RNA chains can still bind rifampicin, although the enzyme activity is not inhibited by such binding. The values of overall binding constants calculated from the kinetic parameters, 1-2 × 10-9 M, are in good agreement with the values of the apparent Kd obtained from fluorimetric titrations and K, determined by enzymatic assays. In addition, the observations that the formation of an initiation complex leads to a significant but not complete rifampicinresistant RNA synthesis and the recent finding that rifampicin only partly inhibits the formation of the first phosphodiester bond in an abortive initiation of RNA chains are consistent with our kinetic mechanism, i.e., the existence of two forms of the rifampicin-RNA pojymerase complex, only one of which is able to initiate the RNA chains.

頁(從 - 到)2669-2676
出版狀態Published - 1 六月 1976


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