The role of spermine in preventing misacylation by phenylalanyl-tRNA synthetase.
Physiological concentrations of polyamines contribute significantly to both the speed and precision of aminoacylation of tRNA. Unphysiologically high concentrations of magnesium ion are required to obtain high rates of synthesis of phenylalanyl-tRNAPheyeast catalyzed by yeast phenylalanine:tRNA ligase in vitro. Under such conditions, rates of misacylation (e.g. the synthesis of Phe-tRNAValE.coli) may be one-fifth of the rate of correct acylation. High rates of correct aminoacylation are achieved in the presence of physiological concentrations of magnesium (1.0 mM) plus spermine (0.2 mM). Under these conditions, there is almost no misacylation. A kinetic study of the magnesium dependence of aminoacylation shows that the rate-determining transition state for Phe-tRNAPhe synthesis contains, in addition to tightly bound Mg2+, either two spermines (Kd = approximately 50 microM) or 2 Mg2+ ions (Kd = approximately 1.0 mM). We postulate that the tRNAPhe binds two spermines to form a very compact, very precisely defined structure that easily forms an activated E.S conformer, E.S (Jencks' Circe Effect). In the absence of spermine, 2 Mg2+ ions bind somewhat more poorly to the tRNA to form a similar but not identical tRNA conformer which, in turn, is less able to form E.S and thus more slowly aminoacylated. Misacylation of noncognate tRNA requires several additional, more loosely bound Mg2+ ions that serve to relax the tRNA structure. Such magnesium-driven relaxation has a minor effect on Km (tRNA), but the resulting floppy structure is able to avoid the barriers against reaction and is amino-acylated thousands of times more rapidly than the compact defined structure.
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D004926:Escherichia coli; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010652:Phenylalanine-tRNA Ligase / Q000378:metabolism*; D012343:RNA, Transfer; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013045:Species Specificity; D013096:Spermine / Q000494:pharmacology*
D012346:RNA, Transfer, Amino Acyl; D013096:Spermine; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; D010652:Phenylalanine-tRNA Ligase; D008274:Magnesium