Mechanism of Action of Rifamazine, a Member of a New Class of (Dimeric) Rifamycins

Fietta, A.; Silvestri, L. G.

doi:10.1111/j.1432-1033.1975.tb04007.x

Cited by 8 publications

(4 citation statements)

References 36 publications

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“…Similar selectivity was shown for the inhibition by rifampicin of RNA synthesis (Hartmann et al, 1967). However, unlike bis(Ans), rifampicin exerts its effect by binding to a single site on RNA polymerase (di Mauro et al, 1969;Wehrli and Staehelin, 1969), although there are indications that another weak binding site may exist (Riva et al, 1972;Fietta and Sylvestri, 1975). From the fluorimetric titration studies (Figure 7), we have shown that at -5 M, bis(Ans) binds to approximately 16-18 strong sites.…”

Section: Discussionmentioning

confidence: 67%

Inhibition of Escherichia coli RNA polymerase by bis(1-anilino-8-naphthalenesulfonate)

Wu¹,

Wu²

1978

Biochemistry

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Section: Discussionmentioning

confidence: 67%

Inhibition of Escherichia coli RNA polymerase by bis(1-anilino-8-naphthalenesulfonate)

Wu¹,

Wu²

1978

Biochemistry

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“…Since it has been reported that a second rifampicin binding site exists on RNA polymerase Fietta and Sylvestri, 1975), a question may be raised whether our kinetic results can be influenced by the binding of a second rifampicin molecule at higher concentrations. We have, therefore, determined the stoichiometry of binding of [14C] rifampicin to the enzyme using both equilibrium dialysis and gel filtrations.…”

Section: Stoichiometry Of Rifampicin Bindingmentioning

confidence: 97%

“…While the values of dissociation constant, K\ = (k-\jk\), for T -Lhe antibiotic rifamycin inhibits bacterial RNA synthesis (Hartmann et al 1967) by binding to the DNA-dependent RNA polymerase (Wehrli et al, 1968;di Mauro et al, 1969;. The interaction of RNA polymerase with rifamycin has been extensively studied: (1) Rifamycin binds tightly to a single site on both the holo and core enzyme by a noncovalent interaction (di Mauro et al, 1969;Wehrli and Staehelin, 1969); however, there are indications that another weak binding site may exist Fietta and Sylvestri, 1975). (2) Genetic evidence suggests that the rifamycin binding site resides in the /3 subunit (Heil and Zillig, 1970).…”

mentioning

confidence: 99%

Molecular mechanism of the rifampicin-RNA polymerase interaction

Yarbrough¹,

Wu²,

Wu³

1976

Biochemistry

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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 less than or equal to 3 x 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: (See article). 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, decrease in the order of core enzyme greater than holoenzyme greater than 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 rifampicin-induced isomerization (conformational change) of the enzyme. This is supported by our observations 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 x 10(-9) M, are in good agreement with the values of the apparent Kd obtained from fluorimetric titrations and Ki determined by enzymatic assays. In addition, the observations that the formation of an initiation complex leads to a significant but not complete rifampicin-resistant 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 mechansim, i.e., the existence of two forms of the rifampicin-RNA polymerase complex, only one of which is able to initiate the RNA chains.

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“…It has been shown by Silvestri et al [33] that RNA polymerase is inhibited by a dimeric derivative of rifamycin SV. Employing ultracentrifugation they failed to demonstrate the existence of a ternary complex enzyme .…”

Section: The Interaction Of R N a Polymerase With Dimeric Derivativesmentioning

confidence: 99%

Studies of the Topography of the Binding Site of DNA-Dependent RNA Polymerase from Escherichia coli for the Antibiotic Rifamycin SV

Stender

Scheit

1977

Eur J Biochem

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The synthesis of dimeric derivatives of rifamycin SV differing in the length of spacer, of derivatives of rifamycin SV possessing 2,4-dinitrophenyl groups in varying distances relative to the aromatic part of the antibiotic and a derivative of rifamycin SV carrying a biotinyl residue is described. Rifamycin SV covalently attached to bovine serum albumin was employed to produce antibodies against rifamycin SV in rabbits. Rifamycin SV as well as 2,4-dinitrophenyl-specific antibodies and avidin were used to study the interaction of RNA polymerase with the respective derivatives of rifamycin SV. The results of this investigation can be summarized as follows.1. Antibodies, which are specific for rifamycin SV do not recognize the rifamycin SV molecule in the enzyme . antibiotic complex.2. 2,4-Dinitrophenyl-specific antibodies are unable to recognize the 2,4-dinitrophenyl residue in the complex enzyme . 2,4-dinitrophenylaminoethylthio-rifamycin-SV.3. However, 2,4-dinitrophenyl groups in complexes between enzyme and 2,4-dinitrophenyl derivatives of rifamycin SV are recognized, if those are separated far enough from the rifamycin part.4. RNA polymerase binds to the rifamycin SV portion in complexes avidin . biotinylaminoethylthio-rifamycin-SV.5. Dimeric rifamycin SV molecules do not form ternary complexes with RNA polymerase. From those results it is concluded that the binding site of RNA polymerase for rifamycin SV extends 1.40 -1.90 nm deep into the interior of the enzyme structure and that the ansa chain of the antibiotic extends furthest into the enzyme Rifamycin SV and its derivaties are strong inhibitors of DNA-dependent RNA synthesis in bacteria [I -51. They interfere with chain initiation of RNA synthesis when binding to the enzyme, RNA polymerase, or to the complex RNA-polymerase . DNA [6,7]. Recent investigations yield evidence that the first phosphodiester bond is still formed in the presence of the antibiotic and that the steps of the enzymatic reaction, which lead to the elongation of the phosphodiester bond, are inhibited [8]. Kinetic and thermodynamic studies reveal the high affinity of rifampicin and other rifamycin SV derivatives to enzyme or enzyme . DNA complexes [9-121. Little is known about the topology of the antibiotic binding site of Abbreviations. The abbreviations of the various synthetic derivatives of rifamycin SV are given in Table 2; Nzph = dinitrophenyl.Enzyme. DNA-dependent RNA polymerase (EC 2.7.7.6).RNA polymerase. An investigation of a rifampicininsensitive RNA polymerase from Escherichia coli and affinity labelling studies with chemically reactivederivatives of rifamycin SV demonstrate that the p subunit seems to carry the antibiotic binding site [13,14]. The affinity labelling experiments indicate that the bound rifamycin SV is in contact with o subunit. Structure-activity relations based on chemical modifications of the antibiotic affecting the activity indicate that both the ansa ring, as well as the aromatic part of the antibiotic, are involved in the interaction with the enzyme (F...

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Mechanism of Action of Rifamazine, a Member of a New Class of (Dimeric) Rifamycins

Cited by 8 publications

References 36 publications

Inhibition of Escherichia coli RNA polymerase by bis(1-anilino-8-naphthalenesulfonate)

Inhibition of Escherichia coli RNA polymerase by bis(1-anilino-8-naphthalenesulfonate)

Molecular mechanism of the rifampicin-RNA polymerase interaction

Studies of the Topography of the Binding Site of DNA-Dependent RNA Polymerase from Escherichia coli for the Antibiotic Rifamycin SV

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