Phenylalanyl-tRNA synthetases from sheep liver and yeast. Correlation between net charge and binding to ribosomes.
Unlike phenylalanyl-tRNA synthetase from lower eukaryotes, the corresponding enzyme from higher eukaryotes displays a pronounced tendency to associate with ribosomes in vitro. To attempt to uncover the structural features responsible for this difference in behavior, a comparative study of the enzymes purified to homogeneity from sheep liver and yeast was undertaken. The two alpha 2 beta 2-type enzymes displayed remarkably similar subunit molecular masses (71 and 63 kDa for sheep, 74 and 63 kDa for yeast), yet differed markedly in their isoelectric points (8.0 and 5.6 pH units, respectively). Mild tryptic digestion of the enzyme from sheep led to preferential degradation of the 63-kDa beta subunit into two major fragments of 35 and 25 kDa, respectively, with concomitant loss of activity. The isoelectric points of the denatured fragments were found to be distinctly lower than that of the denatured beta subunit, implying that the residues responsible for the basic net charge of the original beta subunit are mainly clustered in a small portion of the polypeptide chain which was excised during proteolysis. Despite their different isoelectric points, the enzymes from yeast and sheep displayed identical requirements for aminoacylation of tRNA at optimal rates. Moreover, the incidence of variations in pH and ionic strength on the kinetic parameters of the two enzymes was indistinguishable. Interpreted in terms of the polyelectrolyte theory, these results support the view that the residues responsible for the basic net charge of the mammalian enzyme are located in a region distal from the active site. It is suggested that the cationic charge of the enzyme allows anchorage to a cellular component carrying negative charges, possibly the ribosome.
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D000818:Animals; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D010652:Phenylalanine-tRNA Ligase / Q000302:isolation & purification / Q000378:metabolism*; D012270:Ribosomes / Q000201:enzymology*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012756:Sheep; D013045:Species Specificity
D046911:Macromolecular Substances; D000604:Amino Acyl-tRNA Synthetases; D010652:Phenylalanine-tRNA Ligase