NEW ASPECTS OF THE KINETICS OF INHIBITION BY LINCOMYCIN OF PEPTIDE-BOND FORMATION

We have investigated the inhibition of peptide bond formation by the a ntibiotic lincomycin, at 150 mM NH4Cl. We have used an in vitro system in which a ribosomal ternary complex, the acetyl[H-3] phenytalanine-t RNA-70 S ribosome-poly(U) complex (complex C), reacts with puromycin, forming peptide bonds. Complex C can be considered an analog of the el ongating ribosomal complex and puromycin an analog of aminoacyl-tRNA. in a previous study we reported on the kinetics of inhibition by linco mycin at 100 mM NH4Cl. In the present investigation, we find that an i ncrease of the ammonium ion concentration to 150 mM causes profound ch anges in the kinetic behavior of the system, which can be summarized a s follows. First, the association rate for complex C and lincomycin is increased. At a lincomycin concentration of 10 mu M the apparent equi libration rate constant is 4.3 min(-1) at 100 mM NH4Cl, whereas it bec omes 6.7 min(-1) at 150 mM. Second, at 150 mM NH4Cl, with increasing c oncentrations of lincomycin, there is a transition from competitive to mixed-noncompetitive inhibition. The prevailing notion is that lincom ycin acts at the ribosomal A-site, a mechanism that agrees only with c ompetitive kinetics (mutually exclusive binding between puromycin and lincomycin). At the molecular level, the change in the kinetics of inh ibition that we observe may mean that the mutually exclusive binding b etween aminoacyl-tRNA and lincomycin is converted to simultaneous bind ing, as a result of conformational changes occurring in the elongating ribosomal complex.