Structure/function correlation of spermine‐analogue‐induced modulation of peptidyltransferase activity

Abstract

In a cell-free system derived from Escherichia coli,various analogues of spermine were used to study their effect on the binding of AcPhe-tRNA to poly (U)-programmed ribosomes and on the puromycin reaction carried out at 6 mM Mg 2ϩ (Ac, acetyl). In the absence of factors washable from ribosomes (FWR fraction), mono-acylated or di-acylated analogues of spermine stimulate the binding of AcPhe-tRNA to a lesser degree than spermine, in the order: N 1 -acetylspermine Ͼ N 1 ,N 12 -diacetylspermine Х N 1 ,N 12 -dipivaloylspermine. Also, the above analogues do not show any sparing effect on Mg 2ϩ requirements for AcPhe-tRNA binding to ribosomes, in contrast to spermine. The presence of FWR fraction during the binding or acetylation of the secondary amines of spermine moderates or abolishes the stimulatory effect.In addition, all analogues tested enhance the stability of the ternary complex AcPhe-tRNA-poly(U)-ribosome and the extent of AcPhe-puromycin synthesis, particularly in the absence of the FWR fraction. At the kinetic phase of AcPhe-puromycin synthesis, the analogues display both stimulatory and inhibitory effects, depending on the absence (partial noncompetitive inhibition) or the presence of the FWR fraction (nonessential activation in concert with partial noncompetitive inhibition). Detailed kinetic analysis shows that the analogues tested can mimic the behaviour of spermine, however, the potency to affect the peptidyltransferase activity depends on their degree of acylation, acyl-substituent size, charge distribution and on their chain flexibility.Keywords : acylated polyamine; protein synthesis; puromycin reaction; peptidyltransferase.Polyamines are organic polycations that are protonated at physiological pH and can potentially interact with a variety of cellular macromolecules including nucleic acids and proteins [1]. Substantial evidence exists suggesting a significant role of polyamines in regulating the translation process at several levels [1]. Due to its four positive charges, spermine is the most effective of the naturally occurring polyamines both in regulating the ribosomal functions and in decreasing the Mg 2ϩ requirements for protein synthesis. Tabor and Tabor [2] have reported that endogenous spermine does not exist in Escherichia coli. However, small amounts of spermine, about two orders of magnitude lower than those of spermidine, can be detected in E. coli by more sensitive methods [3]. Extensive biochemical studies have focused on the mechanism of spermine biological action in prokaryotic-cell-free systems, since this polyamine consists the parent compound of a large number of synthetic polyamine analogues with antiparasitic and antitumor activity [4Ϫ6].Evidently, spermine markedly stimulates the activity of several in vitro protein-synthesizing systems characterized by low Mg 2ϩ concentrations near those observed in vivo [7Ϫ10]. In a recent report [11], we have demonstrated that, at 6 mM Mg 2ϩ spermine exhibits a concentration-dependent allosteric biphasic

Unraveling New Features of Clindamycin Interaction with Functional Ribosomes and Dependence of the Drug Potency on Polyamines

“…2, clindamycin hardly affects the binding of AcPhe-tRNA to both sites of poly(U)-programmed ribosomes if the binding is performed in the absence of polyamines; although a small increase in binding is visible in the presence of clindamycin, the differences from the control values are not statistically significant ( p Ͻ 0.05). In accordance with previous results (24,33), spermine or a mixture of spermine and spermidine stimulates the AcPhe-tRNA binding and enhances the stability of the formed 70 S initiation complex. The addition of clindamycin into the polyamine buffer improves further the AcPhe-tRNA binding to both sites but impairs the stability of the initiation ribosomal complex formed.…”

“…In agreement with previous results obtained at 6 mM Mg 2ϩ (30,33), the addition of spermine at this concentration increases the k 3 value of PTase without affecting the K s dissociation constant (Table 1). Consequently, the ratio k 3 / K s expressing the activity status of PTase is enhanced by 43%.…”

Molecular Characterization of Ancylostoma Inhibitors of Coagulation Factor Xa

“…The DYNAFIT method for model discrimination predicted a complex mechanism, in which the inhibitors bind at two separate binding sites, whereas the resulting E⅐I 2 complex retains catalytic activity. Similar inhibitory mechanisms have been described for other hydrolytic enzymes, including D-fructose-1,6-bisphosphate 1-phosphohydrolase (40), ribosomal peptidyltransferase (41), and 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (42).…”

Effects of Ethyl and Benzyl Analogues of Spermine on Escherichia coli Peptidyltransferase Activity, Polyamine Transport, and Cellular Growth

“…Partially purified translation factors (FWR fraction) and crude acetyl-(Ac) [ 3 H]Phe-tRNA charged with 16.3 pmol of [ 3 H]Phe (86 kcpm total) per A 260 unit were prepared as reported previously (15). Complex C, i.e., the Ac[ 3 H]Phe-tRNA-poly(U)-ribosome complex, was prepared and purified through adsorption on cellulose nitrate filters, as described elsewhere (18). The radioactivity trapped on the filters was counted in a liquid scintillation spectrometer.…”

“…The amino acid residues in PotD and PotF involved in the interaction with polyamines have been revealed by mutational and x-ray analysis (20,37,40). Recently, we have studied in E. coli the interactions of acetyl polyamines with their transporters (17) or peptidyltransferase (18), and we have evaluated the significance of polyamine primary and secondary amino groups, as well as that of chain flexibility, as determinants of these bacterial functions. Since acetyl polyamines per se have no pharmaceutical significance, it was of interest to extend our knowledge by using a series of spermine analogues which are known to have an antiproliferative effect on eukaryotic cells.…”