Abstract
We have developed anin vitrosystem for the determination of peptidyltransferase activity in rabbit reticulocyte ribosomes. Using this system, a detailed kinetic analysis of a model reaction for peptidyltransferase is described, with AcPhe-tRNA as the peptidyl donor and puromycin as the acceptor. The [AcPhe-tRNA–poly(U)–80S ribosome] complex (complex C) is isolated and then reacted with excess puromycin to give AcPhe-puromycin. This reaction (puromycin reaction) follows first-order kinetics at all concentrations of puromycin tested. At saturating concentrations of puromycin, the first-order rate (k3) constant is identical to the catalytic rate constant (kcat) of peptidyltransferase. Thisk3of peptidyltransferase is equal to 2.9 min−1at 37°C. Moreover, the ratiok3/Ks, which is an accurate measure of peptidyltransferase activity, was increased 80-fold when salt-washed ribosomes were replaced by unwashed ribosomes. Finally, the puromycin reaction was inhibited by several well-known antibiotics acting on the eukaryotic peptidyltransferase.
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