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 Cl, which is inactive toward puromycin. Cl is formed via a two-step mechanism in which Cl is the product of a slow conformational change of the initial encounter complex Cl according to the equation C + l reversible Cl reversible Cl. Furthermore, we provide evidence that analog 5 may react further with Cl forming the species Cl2. 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.