Type of inhibition of peptide bond formation by chloramphenicol depends on the temperature and the concentration of ammonium ions.
Using the same system that we used in a previous study [Eur. J. Biochem. 164:53-58 (1987)], we have further examined the kinetics of inhibition of peptide bond formation by chloramphenicol in the puromycin reaction and we have applied conditions that are known to cause conformational changes to the 70 S ribosome. These conditions are the change in reaction temperature from 25 degrees to 5 degrees and the change in the concentration of NH4+ ion (50 mM versus 100 mM). The initial transient phase of competitive inhibition is now (100 mM NH4+ and 5 degrees or 50 mM NH4+ and 25 degrees) much more pronounced than at 100 mM NH4+ and 25 degrees. Simple competitive inhibition is the only type of inhibition we can find when analyzing the kinetic information given by the initial slopes of the first-order time plots. This contrasts with the kinetics observed at 100 mM NH4+ and 25 degrees, where a transient phase of competitive inhibition is followed (at higher concentrations of chloramphenicol) by a phase of mixed noncompetitive inhibition, which corresponds to a lower kcat for peptidyltransferase (EC 2.3.2.12). This pattern of inhibition (competitive-mixed noncompetitive) was again obtained in this study using a ribosomal complex [acetyl[3H]Phe-tRNA-poly(U)-ribosome] of low peptidyltransferase activity (kcat = 0.91 min-1), as was obtained previously when we used a complex of high activity (kcat = 2.00 min-1). Thus, the lowering of the kcat of peptidyltransferase induced by chloramphenicol (from 0.91 to 0.34 min-1) can occur irrespective of the activity status of peptidyltransferase. The conformational changes that are induced by chloramphenicol and lead to the lowering of the kcat of peptidyltransferase need both relatively high (100 mM) concentrations of monovalent ion and higher temperature (25 degrees as opposed to 5 degrees). If these conditions are not met, the inhibition is simple competitive and the kcat of peptidyltransferase remains unchanged. These results offer an explanation as to why a clear-cut competitive inhibition of the puromycin reaction by chloramphenicol has been difficult to observe for so many years.
Kalpaxis
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DL
Laboratory of Biochemistry, School of Medicine, University of Patras, Greece.
Coutsogeorgopoulos
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C
D000641:Ammonia / Q000494:pharmacology*; D002414:Cations, Monovalent; D002701:Chloramphenicol / Q000494:pharmacology*; D004926:Escherichia coli; D066298:In Vitro Techniques; D007700:Kinetics; D010441:Peptide Chain Elongation, Translational / Q000187:drug effects*; D011691:Puromycin / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D013696:Temperature
D002414:Cations, Monovalent; D011691:Puromycin; D002701:Chloramphenicol; D000641:Ammonia