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

In a cell-free system derived from Escherichia coli, lincomycin produces bi phasic logarithmic time plots for inhibition of peptide-bond formation when puromycin is used as an acceptor substrate and AcPhe-tRNA as a donor subst rate. In a previous study, initial slope analysis of the logarithmic time p lots revealed that the encounter complex CI between the initiator ribosomal complex (C) and lincomycin (I) undergoes a slow isomerization to C*I. Duri ng this change, the bound AcPhe-tRNA and lincomycin are rearranged to also accommodate puromycin, and this may account for the mixed noncompetitive in hibition (K-i* = 70 mu M) 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 w ith a second molecule of I, giving C*I-2 complex. However, the logarithmic time plots remain biphasic even at high concentrations of lincomycin, makin g possible the identification of another inhibition constant K-i*', which i s equal to 18 mu M. The simplest explanation of this finding is to assume t he existence of a second isomerization step C*I-2 reversible arrow 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 u ndergoes a slow isomerization, which may be the result of conformational ch anges induced by the drug.