Slow sequential conformational changes in Escherichia coli ribosomes induced by lincomycin: kinetic evidence

Mol Pharmacol. 1999 Nov;56(5):1042-6. doi: 10.1124/mol.56.5.1042.

Abstract

In a cell-free system derived from Escherichia coli, lincomycin produces biphasic logarithmic time plots for inhibition of peptide-bond formation when puromycin is used as an acceptor substrate and AcPhe-tRNA as a donor substrate. In a previous study, initial slope analysis of the logarithmic time plots revealed that the encounter complex CI between the initiator ribosomal complex (C) and lincomycin (I) undergoes a slow isomerization to C*I. During this change, the bound AcPhe-tRNA and lincomycin are rearranged to also accommodate puromycin, and this may account for the mixed noncompetitive inhibition (K(i)* = 70 microM) established at higher concentrations of the drug. The above-mentioned effect was further investigated by analyzing the late phase of the logarithmic time plots. It was found that C*I complex reacts with a second molecule of I, giving C*I(2) complex. However, the logarithmic time plots remain biphasic even at high concentrations of lincomycin, making possible the identification of another inhibition constant K(i)*', which is equal to 18 microM. The simplest explanation of this finding is to assume the existence of a second isomerization step C*I(2) <--> C*I(2'), slowly equilibrated. The determination of K(i)*' enables us to calculate the isomerization constant (K(isom) = 2.9) with the formula K(i)*' = K(i)*/(1 + K(isom)). Our results suggest that whenever a fast and reversible interaction of lincomycin with the elongating ribosomal complex C occurs, the latter undergoes a slow isomerization, which may be the result of conformational changes induced by the drug.

MeSH terms

  • Anti-Bacterial Agents / pharmacology*
  • Enzyme Inhibitors / pharmacology
  • Escherichia coli / chemistry
  • Escherichia coli / drug effects*
  • Kinetics
  • Lincomycin / pharmacology*
  • Macromolecular Substances
  • Peptidyl Transferases / antagonists & inhibitors*
  • Ribosomes / drug effects*
  • Ribosomes / ultrastructure

Substances

  • Anti-Bacterial Agents
  • Enzyme Inhibitors
  • Macromolecular Substances
  • Lincomycin
  • Peptidyl Transferases