Inhibition of ribosomal peptidyltransferase by chloramphenicol. Kinetic studies.

D Drainas, D L Kalpaxis, C Coutsogeorgopoulos

European journal of biochemistry / FEBS 1987 Apr 1

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The mechanism of action of chloramphenicol in inhibiting peptide bond formation has been examined with the aim of discovering whether chloramphenicol brings about conformational changes in the peptidyltransferase domain, its target locus on the ribosome. These conformational changes have been sought as changes in the catalytic rate constant of peptidyltransferase. A detailed kinetic analysis of the inhibition of the puromycin reaction in a system derived from Escherichia coli [Kalpaxis et al. (1986) Eur. J. Biochem. 154, 267-271] has been carried out. There is an initial phase of competitive inhibition (Ki = 0.7 microM) in which the double-reciprocal plots are linear. This phase is observed at concentrations of chloramphenicol up to about 3.0 microM (4.3 Ki). By increasing the concentration of the inhibitor the kinetics change and the inhibition becomes no longer of the competitive type. These results are obtained when the inhibitor is added simultaneously with the substrate (puromycin). Preincubation with the inhibitor before the addition of puromycin gives hyperbolic double-reciprocal plots at inhibitor concentrations around the Ki. After preincubation with the inhibitor at concentrations above the Ki (3-100 Ki) the double-reciprocal plots are linear again and indicate complete, mixed non-competitive inhibition. Analogous behaviour is observed with thiamphenicol (Ki = 0.45 microM) and tevenel (Ki = 1.7 microM). It is proposed that initially chloramphenicol and its two analogs interact with puromycin at a ribosomal locus (peptidyltransferase domain) in a mutually exclusive binding mode (competitive kinetics). Soon after this initial interaction, the antibiotic induces conformational changes to the peptidyltransferase domain so that puromycin is accepted and peptide bonds are still formed but with a lower catalytic rate constant. At this latter state, the ribosome can accept both the inhibitor and the substrate (puromycin) but then, if the concentration of the inhibitor is sufficiently high, peptide bonds are not formed (complete, linear mixed non-competitive inhibition).

Citation

D Drainas, D L Kalpaxis, C Coutsogeorgopoulos. Inhibition of ribosomal peptidyltransferase by chloramphenicol. Kinetic studies. European journal of biochemistry / FEBS. 1987 Apr 1;164(1):53-8