New aspects of the kinetics of inhibition by lincomycin of peptide bond formation.
We have investigated the inhibition of peptide bond formation by the antibiotic lincomycin, at 150 mM NH4Cl. We have used an in vitro system in which a ribosomal ternary complex, the acetyl[3H] phenylalanine-tRNA-70 S ribosome-poly(U) complex (complex C), reacts with puromycin, forming peptide bonds. Complex C can be considered an analog of the elongating ribosomal complex and puromycin an analog of aminoacyl-tRNA. In a previous study we reported on the kinetics of inhibition by lincomycin at 100 mM NH4Cl. In the present investigation, we find that an increase of the ammonium ion concentration to 150 mM causes profound changes in the kinetic behavior of the system, which can be summarized as follows. First, the association rate for complex C and lincomycin is increased. At a lincomycin concentration of 10 microM the apparent equilibration rate constant is 4.3 min-1 at 100 mM NH4Cl, whereas it becomes 6.7 min-1 at 150 mM. Second, at 150 mM NH4Cl, with increasing concentrations of lincomycin, there is a transition from competitive to mixed-noncompetitive inhibition. The prevailing notion is that lincomycin acts at the ribosomal A-site, a mechanism that agrees only with competitive kinetics (mutually exclusive binding between puromycin and lincomycin). At the molecular level, the change in the kinetics of inhibition that we observe may mean that the mutually exclusive binding between aminoacyl-tRNA and lincomycin is converted to simultaneous binding, as a result of conformational changes occurring in the elongating ribosomal complex.
Kallia-Raftopoulos
S
S
Laboratory of Biochemistry, School of Medicine, University of Patras, Greece.
Kalpaxis
D L
DL
Coutsogeorgopoulos
C
C
D001667:Binding, Competitive; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008034:Lincomycin / Q000378:metabolism / Q000494:pharmacology*; D008956:Models, Chemical; D010441:Peptide Chain Elongation, Translational / Q000187:drug effects*; D011691:Puromycin / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism
D012346:RNA, Transfer, Amino Acyl; C006383:tRNA, N-acetylphenylalanine-; D011691:Puromycin; D008034:Lincomycin