KINETIC-STUDIES ON THE ACTIVATION OF EUKARYOTIC PEPTIDYLTRANSFERASE BY POTASSIUM

In an effort to elucidate the role of potassium ions in the formation of peptide bond, we have used the reaction between puromycin and a rib osomal complex (from rabbit reticulocytes) bearing the donor substrate , AcPhe-tRNA, prebound at the so called P site (puromycin-reactive sta te). This reaction can be analyzed as a first-order reaction. At satur ating concentrations of puromycin (S) the first-order rate constant (k (max)(S)) is a measure of the apparent catalytic rate constant of pept idyltransferase in the puromycin reaction. This k(max)(S) depends on t he concentration of potassium ions and increases when the concentratio n of K+ is increased. The data suggest a kinetic model in which potass ium acts as an essential activator in the puromycin reaction. A single molecule of potassium participates in the mechanism of activation. Th e kinetics correspond to a sequential addition of potassium and puromy cin to two separate and independent sites on the ribosome. At saturati ng levels of both K+ and S the maximal value for the catalytic rate co nstant of peptidyltransferase (k(p)) is equal to 20 min(-1) at 25 degr ees C. (C) 1997 Academic Press.