Vol. 56, Issue 5, 1042-1046, November 1999

Slow Sequential Conformational Changes in Escherichia coli Ribosomes Induced by Lincomycin: Kinetic Evidence

Sofia Kallia-Raftopoulos and Dimitrios L. Kalpaxis

Laboratory of Biochemistry, School of Medicine, University of Patras, Patras, Greece

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 constant K_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.