DETERMINATION OF EUKARYOTIC PEPTIDYLTRANSFERASE ACTIVITY BY PSEUDO-FIRST-ORDER KINETIC-ANALYSIS

We have developed an in vitro system for the determination of peptidyl transferase activity in rabbit reticulocyte ribosomes. Using this syst em, a detailed kinetic analysis of a model reaction for peptidyltransf erase is described, with AcPhe-tRNA as the peptidyl donor and puromyci n as the acceptor. The [AcPhe-tRNA-poly(U)-80S ribosome] complex (comp lex C) is isolated and then reacted with excess puromycin to give AcPh e-puromycin. This reaction (puromycin reaction) follows first-order ki netics at all concentrations of puromycin tested. At saturating concen trations of puromycin, the first-order rate (k(3)) constant is identic al to the catalytic rate constant (h(cat)) of peptidyltransferase. Thi s k(3) of peptidyltransferase is equal to 2.9 min(-1) at 37 degrees C. Moreover, the ratio k(3)/K-s, which is an accurate measure of peptidy ltransferase activity, was increased 80-fold when salt-washed ribosome s were replaced by unwashed ribosomes. Finally, the puromycin reaction was inhibited by several well-known antibiotics acting on the eukaryo tic peptidyltransferase. (C) 1997 Academic Press.