In a cell-free system derived from Escherichiu coli, the reaction between A c [~H ] P~~-~R N A and puromycin (S) is inhibited by blasticidin S (I). In this reaction A c [~H ] P~~-~R N Ais part of the A~[~HlPhe-tRNA-poly(U)-ribosome complex (C). After preincubating the complex C with I and then adding S, the degree of inhibition is greater than that observed when C reacts with a mixture of S and I. Without preincubation, the inhibition is competitive giving a Ki of 2 x M. After preincubation the inhibition becomes of the mixed non-competitive type. A first-order kinetic analysis of the reaction between C and excess S, in the presence or in the absence of I, with or without preincubation, suggests that I acts as a modifier decreasing the catalytic rate constant of ribosomal peptidyltransferase (the putative enzyme that catalyzes the reaction between C and S). The effectiveness of I cannot be expressed by an equilibrium constant such as the above-mentioned Ki. A model is proposed which explains the results obtained. In this model, in the presence of I, C is converted to a modified species C *, which is still able to react with S but with a lower catalytic rate constant. This is a novel concept, in which the ribosome can be subjected to modulation of its activity by small ligands. It can be useful in studies on translational control of protein synthesis.It has been widely assumed that the interaction between the ribosome (R) and various inhibitors (I) of protein synthesis can be expressed by a simple equilibrium of the form R + I R1, and that the corresponding equilibrium constant (K,) is a measure of the potency of the inhibitor. This notion has prevailed for many years in most of the studies on inhibitors of protein synthesis. In the field of ribosomal topography attempts are made to map ribosomal sites based, again, on the assumption of simple equilibria [I, 21. The first indications that a simple equilibrium might express only the initial encounter between the ribosome and the inhibitor, and that subsequent events may lead to a modification of the ribosome, came from work with sparsomycin and spiramycin [3], which was confirmed in other laboratories [4, 51. Blasticidin S is an aminohexose pyrimidine nucleoside antibiotic, which inhibits protein synthesis. It specifically inhibits ribosomal peptidyltransferase [6, 71. The interest in blasticidin S is due to the fact that structurally it relates to puromycin and to the aminoacyladenylyl terminus of aminoacyl-tRNA; it has been used in studies involving other important inhibitors of protein synthesis, such as chloramphenicol [8 -101 and sparsomycin [I 11. However, the mechanism of action of blasticidin S, as well as that of chloramphenicol and of sparsomycin is still unclear. Blasticidin s has also been reported as an inhibitor of DNA synthesis [12].A model reaction, used frequently in studies on protein synthesis, is the puromycin reaction. Puromycin is utilized as Abbreviation. Complex C, the AcPhe-tRNA -poly(U) -ribosome complex. a substrate by the ribosomal pept...
The mechanism of action of chloramphenicol in inhibiting peptide bond formation has been examined with the aim of discovering whether chloramphenicol brings about conformational changes in the peptidyltransferase domain, its target locus on the ribosome. These conformational changes have been sought as changes in the catalytic rate constant of peptidyltransferase. A detailed kinetic analysis of the inhibition of the puromycin reaction in a system derived from Escherichia coli [Kalpaxis et al. (1986) Eur. J. Biochem. 151,267-2711 has been carried out. There is an initial phase of competitive inhibition (Ki = 0.7 pM) in which the double-reciprocal plots are linear. This phase is observed at concentrations of chloramphenicol up to about 3.0 pM (4.3 Ki). By increasing the concentration of the inhibitor the kinetics change and the inhibition becomes no longer of the competitive type. These results are obtained when the inhibitor is added simultaneously with the substrate (puromycin). Preincubation with the inhibitor before the addition of puromycin gives hyperbolic double-reciprocal plots at inhibitor concentrations around the Ki. After preincubation with the inhibitor at concentrations above the Ki (3 -100 Ki) the double-reciprocal plots are linear again and indicate complete, mixed non-competitive inhibition.Analogous behaviour is observed with thiamphenicol (Ki = 0.45 pM) and tevenel (Ki = 1.7 pM). It is proposed that initially chloramphenicol and its two analogs interact with puromycin at a ribosomal locus (peptidyltransferase domain) in a mutually exclusive binding mode (competitive kinetics). Soon after this initial interaction, the antibiotic induces conformational changes to the peptidyltransferase domain so that puromycin is accepted and peptide bonds are still formed but with a lower catalytic rate constant. At this latter state, the ribosome can accept both the inhibitor and the substrate (puromycin) but then, if the concentration of the inhibitor is sufficiently high, peptide bonds are not formed (complete, linear mixed non-competitive inhibition).
The inhibition of peptide bond formation by spiramycin was studied in an in vitro system derived from Escherichia coli. Peptide bonds are formed between puromycin (S) and Ac-Phe-tRNA, which is a component of complex C, i.e., of the [Ac-Phe-tRNA-70S ribosome-poly(U)] complex, according to the puromycin reaction: C+S (Ks)<==>CS (k3)==>C'+P [Synetos, D., & Coutsogeorgopoulos, C. (1987) Biochim. Biophys. Acta 923, 275-285]. It is shown that spiramycin (A) reacts with complex C and forms the spiramycin complex C*A, which is inactive toward puromycin. C*A is the tightest complex formed between complex C and any of a number of antibiotics, such as chloramphenicol, blasticidin S, lincomycin, or sparsomycin. C*A remains stable following gel chromatography on Sephadex G-200 and sucrose gradient ultracentrifugation. Detailed kinetic study suggests that C*A is formed in a variation of a two-step mechanism in which the initial encounter complex CA is kinetically insignificant and C*A is the product of a conformational change of complex CA according to the equation, C+A (kassoc)<==>(kdissoc) C*A. The rate constants of this reaction (spiramycin reaction) are kassoc = 3.0 x 10(4) M-1 s-1 and kdissoc = 5.0 x 10(-5) s-1. Such values allow the classification of spiramycin as a slow-binding, slowly reversible inhibitor; they also lead to the calculation of an apparent overall dissociation constant equal to 1.8 nM for the C*A complex. Furthermore, they render spiramycin a useful tool in the study of antibiotic action on protein synthesis in vitro. Thus, the spiramycin reaction, in conjunction with the puromycin reaction, is applied (i) to detect a strong preincubation effect exerted by chloramphenicol and lincomycin (this effect constitutes further evidence that these two antibiotics combine with complex C as slow-binding inhibitors) and (ii) to determine the rate constant for the regeneration (k7 = 2.0 x 10(-3) s-1) of complex C from the sparsomycin complex C*I [Theocharis, D. A., & Coutsogeorgopoulos, C. (1992) Biochemistry 31, 5861-5868] according to the equation, C+I (Ki)<==>CI (k6)<==>(k7) C*I. The determination of k7 enables us to calculate the apparent association rate constant of sparsomycin, (k7/Ki') = 1.0 x 10(5) M-1 s-1, where Ki' = Ki(k7/k6 + k7). It is also shown that Ac-Phe-tRNA bound to the sparsomycin complex C*I is protected against attack by hydroxylamine.(ABSTRACT TRUNCATED AT 400 WORDS)
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