NEW ASPECTS ON THE KINETICS OF ACTIVATION OF RIBOSOMAL PEPTIDYLTRANSFERASE-CATALYZED PEPTIDE-BOND FORMATION BY MONOVALENT IONS AND SPERMINE

The effect of NH4+ and K+ ions on the activity of ribosomal peptidyltr ansferase was investigated in a model system derived from Escherichia coli, in which AL Phe-puromycin is produced by a pseudo-first-order re action between the preformed AcPhe-tRNA-poly(U)-ribosome complex (comp lex C) and excess puromycin. Detailed kinetic analysis suggests that b oth NH4+ and K+ ions act as essential activators of peptidyltransferas e by filling randomly, but not cooperatively multiple sites on the rib osome. With respect to the NH4+ effect at 25 degrees C, the values of the molecular interaction coefficient (n), the dissociation constant ( K-A), and the apparent catalytic rate constant (k(max)) of peptidyltra nsferase at saturating levels of NH4+ and puromycin are 1.99, 268.7 nM and 24.8 min(-1), respectively. The stimulation of peptidyltransferas e by K+ ions at 25 degrees C (n = 4.38, K-A = 95.5 mM, k(max) = 9.6 mi n(-1)) is not as marked as that caused by NH4+ ions; Furthermore, it i s evident that NH4+ at high concentration (200 mM) is effective in fil ling regulatory sites of complex C, which are responsible for the modu latory effect of spermine. The combination of NH4+ ions (200 mM) with spermine (300 mu M) produces an additive increase in peptidyltransfera se activity. Taken together, these findings suggest the involvement of two related pathways in the regulation of peptidyltransferase activit y, one mediated by specific monovalent cations and the other mediated by spermine. (C) 1997 Elsevier Science B.V.