Slow sequential conformational changes in Escherichia coli ribosomes induced by lincomycin: kinetic evidence.
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.
Kallia-Raftopoulos
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Laboratory of Biochemistry, School of Medicine, University of Patras, Patras, Greece.
Kalpaxis
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DL
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000737:chemistry / Q000187:drug effects*; D007700:Kinetics; D008034:Lincomycin / Q000494:pharmacology*; D046911:Macromolecular Substances; D010458:Peptidyl Transferases / Q000037:antagonists & inhibitors*; D012270:Ribosomes / Q000187:drug effects* / Q000648:ultrastructure
D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D046911:Macromolecular Substances; D008034:Lincomycin; D010458:Peptidyl Transferases