New aspects on the kinetics of activation of ribosomal peptidyltransferase-catalyzed peptide bond formation by monovalent ions and spermine

Abstract

The effect of NH 4 + and K + ions on the activity of ribosomal peptidyltransferase was investigated in a model system derived from Escherichia coli, in which AcPhe-puromycin is produced by a pseudo-first-order reaction between the preformed AcPhe-tRNA-poly(U)-ribosome complex (complex C) and excess puromycin. Detailed kinetic analysis suggests that both NH 4 + and K + ions act as essential activators of peptidyltransferase by filling randomly, but not cooperatively, multiple sites on the ribosome. With respect to the NH 4 + effect at 25°C, the values of the molecular interaction coefficient (n), the dissociation constant ( K A), and the apparent catalytic rate constant ( k max) of peptidyltransferase at saturating levels of NH 4 + and puromycin are 1.99, 268.7 mM and 24.8 min −1, respectively. The stimulation of peptidyltransferase by K + ions at 25°C ( n=4.38, K A=95.5 mM, k max=9.6 min −1) is not as marked as that caused by NH 4 + ions. Furthermore, it is evident that NH 4 + at high concentration (200 mM) is effective in filling regulatory sites of complex C, which are responsible for the modulatory effect of spermine. The combination of NH 4 + ions (200 mM) with spermine (300 μM) produces an additive increase in peptidyltransferase activity. Taken together, these findings suggest the involvement of two related pathways in the regulation of peptidyltransferase activity, one mediated by specific monovalent cations and the other mediated by spermine.