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-pu romycin is produced in a pseudo-first-order reaction between the prefo rmed 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 k inetic analysis suggests that these analogs (I) react competitively wi th complex C and form the complex Cl which is inactive toward puromyc in. Cl is formed via a two-step mechanism in which C*l is the product of a slow conformational change of the initial encounter complex CI a ccording to the equation C + l reversible arrow Cl reversible arrow C l. Furthermore, we provide evidence that analog 5 may react further wi th Cl forming the species C*l(2). The values of the apparent associat ion rate constant (k(assoc)) are 1.45 x 10(4) M(-1) sec(-1) for 2, 5.5 x 10(3) M(-1) sec(-1) for 3, and 1.8 x 10(3) M(-1) sec(-1) for 4. In the case of analog 5, k(assoc) is a linear function of the inhibitor c oncentration; 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 CA M 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 am ong 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.