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 μM) 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*I₂ complex. However, the logarithmic time plots remain biphasic even at high concentrations of lincomycin, making possible the identification of another inhibition constantK _i*′, which is equal to 18 μM. The simplest explanation of this finding is to assume the existence of a second isomerization step C*I₂ ⇌ C*I₂ ^′, 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.