Determination of eukaryotic peptidyltransferase activity by pseudo-first-order kinetic analysis

We have developed an in vitro system 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. This k3 of peptidyltransferase is equal to 2.9 min-1 at 37 degrees C. Moreover, the ratio k3/ 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.