Kinetic studies on ribosomal peptidyltransferase. The behaviour of the inhibitor blasticidin S

Abstract

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...

Polyamines Affect Diversely the Antibiotic Potency

“…In agreement with a previous study performed at 10 mM Mg 2ϩ and 100 mM NH 4 ϩ (6), analysis of the initial slopes by DR plotting (1/(k obs ) o versus 1/[S]) and slope-replotting (slope of DR plots versus [I]), not shown, confirms that blasticidin S behaves as a competitive inhibitor over a narrow range of inhibitor concentrations ([I] Ͻ 3 ϫ K i ). The K i value found in the present study is approximately two-times higher than that reported in this earlier report (6). In contrast, our analysis of the late slopes revealed a partial-noncompetitive inhibition.…”

“…Consistently, blasticidin S has been found to inhibit the binding of CACCA (Phe) to the A-site of ribosomes (3) and to compete with designated A-site inhibitors (4). In addition, the inhibition of peptidyl-or AcPhe 1 -puromycin synthesis by this antibiotic shows a competitive phase in concert with noncompetitive or mixed-noncompetitive phases (5,6), a fact supporting the notion that blasticidin S influences, at least transiently, the affinity of the A-site. Further support derives from chemical probing studies in Escherichia coli ribosomes (7), suggesting that blasticidin S protects A2439, one of the three nucleosides in domain V of 23 S rRNA, which show altered chemical reactivity on removal of the aminoacyl group from A-site-bound tRNAs (8).…”

“…The apparent association rate constant of binding, (k 6 ϩ k 7 )/K i , equals 1.36 ϫ 10 5 M Ϫ1 s Ϫ1 , a value lower than the upper limit of 10 6 M Ϫ1 s Ϫ1 set for the classification of a drug as a slow binding inhibitor (27). In addition, the k 7 value is less than that measured for the forward rate constant, k 6 . Consequently, our results suggest that a fast association-dissociation equilibrium step (competitive phase) is followed by a slower rearrangement step, which falls within the minute time scale.…”

“…Biochemical Preparations-Salt-washed (0.5 M NH 4 Cl) 70 S ribosomes and partially purified translation factors were obtained from E. coli CAN20 -12E cells as described elsewhere (6). Complex C, i.e.…”

“…24) are presented in Table I. As described previously (26), the individual values of k 6 and k 7 can be calculated by nonlinear regression fitting of kinetic data to Equation 3,…”