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 ribosomal complex (from rabbit reticulocytes) bearing the donor substrate, AcPhe-tRNA, prebound at the so-called P site (puromycin-reactive state). This reaction can be analyzed as a first-order reaction. At saturating concentrations of puromycin (S) the first-order rate constant (k(max)S) is a measure of the apparent catalytic rate constant of peptidyltransferase in the puromycin reaction. This k(max)S depends on the concentration of potassium ions and increases when the concentration of K+ is increased. The data suggest a kinetic model in which potassium acts as an essential activator in the puromycin reaction. A single molecule of potassium participates in the mechanism of activation. The kinetics correspond to a sequential addition of potassium and puromycin to two separate and independent sites on the ribosome. At saturating levels of both K+ and S the maximal value for the catalytic rate constant of peptidyltransferase (k(p)) is equal to 20 min(-1) at 25 degrees C.