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

In a cell-free system derived from Escherichia coli,various analogues of sp ermine 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 Mg2+ (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 > N-1,N-12-diacetylspermine congruent to N-1,N-12- dipivaloy lspermine. Also, the above analogues do not show any sparing effect on Mg2 requirements for AcPhe-tRNA binding to ribosomes, in contrast to spermine. The presence of FWR fraction during the binding or acetylation of the seco ndary 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, pa rticularly in the absence of the FWR fraction. At the kinetic phase of AcPh e-puromycin synthesis, the analogues display both stimulatory and inhibitor y effects, depending on the absence (partial noncompetitive inhibition) or the presence of the FWR fraction (nonessential activation in concert with p artial 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 acyl ation, acyl-substituent size, charge distribution and on their chain flexib ility.