Aminoacyl and Peptidyl Analogs of Chloramphenicol as Slow-Binding Inhibitors of Ribosomal Peptidyltransferase: A New Approach for Evaluating Their Potency

Abstract

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*I, which is inactive toward puromycin. C*I is formed via a two-step mechanism in which C*I is the product of a slow conformational change of the initial encounter complex CI according to the equation C + I ⇌ CI ⇌ C*I. Furthermore, we provide evidence that analog 5 may react further with C*I forming the species C*I₂. The values of the apparent association rate constant (k_assoc) are 1.45 × 10⁴m⁻¹ sec⁻¹ for2, 5.5 × 10³m⁻¹sec⁻¹ for 3, and 1.8 × 10³m⁻¹ sec⁻¹ for 4. 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 × 10²m⁻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 than3 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.