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 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 NH4+ and K+ ions act as essential activators of peptidyltransferase by filling randomly, but not cooperatively, multiple sites on the ribosome. With respect to the NH4+ effect at 25 degrees C. the values of the molecular interaction coefficient (n), the dissociation constant (KA), and the apparent catalytic rate constant (kmax) of peptidyltransferase at saturating levels of NH4+ and puromycin are 1.99, 268.7 mM and 24.8 min(-1), respectively. The stimulation of peptidyltransferase by K+ ions at 25 degrees C (n = 4.38, KA = 95.5 mM, kmax = 9.6 min[-1]) is not as marked as that caused by NH4+ ions. Furthermore, it is evident that NH4+ 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 NH4+ ions (200 mM) with spermine (300 microM) 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.

Aminoacyl and peptidyl analogs of chloramphenicol as slow-binding inhibitors of ribosomal peptidyltransferase: a new approach for evaluating their potency.

In a model system derived from Escherichia coli, acetylphenylalanyl-puromycin is produced in a pseudo-first-order reaction between the preformed acetylphenylalanyl/tRNA/poly(U)/ribosome complex (complex C) and excess puromycin. Two aminoacyl analogs [3, Gly-chloramphenicol (CAM): 4, L-Phe-CAM] and two peptidyl analogs (2, L-Phe-Gly-CAM: 5, Gly-Phe-CAM) of CAM (1) were tested as inhibitors in this reaction. Detailed kinetic analysis suggests that these analogs (I) react competitively with complex C and form the complex C*l, which is inactive toward puromycin. C*l is formed via a two-step mechanism in which C*l is the product of a slow conformational change of the initial encounter complex Cl according to the equation C + l reversible Cl reversible C*l. Furthermore, we provide evidence that analog 5 may react further with C*l forming the species C*l2. The values of the apparent association rate constant (K(assoc)) are 1.42 x microM-1 min-1 for 2, 0.55 x microM-1 min-1 for 3, and 0.18 x microM-1 min-1 for 4 and 0.038 x microM-1 min-1 for 5 [corrected]. In the case of analog 5, K(assoc) is a linear function of the inhibitor concentration; when [I] approaches zero, the K(assoc) value is equal to 3.8 x 10(2) M-1 sec-1. Such values allow the classification of CAM analogs as slow-binding inhibitors. According to K(assoc) values, we could surmise that analog 2 is 2.5-fold more potent than 3 and 8-fold more potent than 4. The relative potency of analog 5 is the lowest among the analogs and is dependent on its concentration. The results are compared with previous data and discussed on the basis of a possible retro-inverso relationship between CAM analogs and puromycin.

Insights into the mechanism of azithromycin interaction with an Escherichia coli functional ribosomal complex.

Azithromycin, a derivative of erythromycin with improved activity against Gram-negative bacteria, exhibits a marginal inhibition effect in a model system derived from Escherichia coli, in which a peptide bond is formed between puromycin and AcPhe-tRNA bound at the P-site of poly(U)-programmed ribosomes. This renders the study of azithromycin interaction with Ac[(3)H]Phe-tRNA. poly(U). 70S ribosome complex (complex C) impossible, if we analyze its effect on peptide bond formation. To overcome this problem, we have used an alternative approach to investigate kinetically the azithromycin interaction with complex C and to compare the azithromycin binding properties with those of erythromycin. This approach was based on the ability of azithromycin to compete with tylosin, a macrolide antibiotic strongly inhibiting the puromycin reaction. Detailed kinetic analysis revealed that the encounter complex CA between complex C and azithromycin (A) undergoes a slow isomerization to a tighter complex C*A, which remains active toward puromycin. The determination of inhibition and isomerization rate constants enabled us to classify azithromycin as a slow-binding ligand of ribosomes. Compared with erythromycin, azithromycin is a better inducer and stabilizer of the C*A complex. This finding may explain the superiority of azithromycin as inhibitor of translation in E. coli cells and many other Gram-negative bacteria.