Tryptophanyl transfer ribonucleic acid synthetase of Escherichia coli. Character of required thiol group and structure of thiol peptides.
Native tryptophanyl-tRNA synthetase purified from Escherichia coli B has on each identical subunit a single thiol group which rapidly forms a mixed disulfide with a thionitrobenzoate moiety of 5,5'-dithiobis(2-nitrobenzoic acid). The reaction and the concomitant inactivation of the enzyme are both reversible by reductive removal of the thionitrobenzoate with dithiothreitol. Iodoacetamide and N-ethylmaleimide also react with the thiol group required for enzyme activity, but iodoacetic acid inactivates the enzyme through another mechanism. Three or 4 half-cystine residues/subunit were detected by amino acid analysis and by titration of the denatured enzyme with 5,5'-dithiobis(2-nitrobenzoic acid); no disulfide bonds were detected by borohydride reduction. Cleavage of the subunit (molecular weight 37,000) with 2-nitro-5-thiocyanobenzoic acid gave fragments of molecular weights 32,000, 27,000, and 9,500. Five carboxymethylated peptides were isolated from the trypsin products of the denatured enzyme after treatment with iodo[14C]lacetate. Three of these peptides represented unique sequences surrounding thiol groups in the enzyme. One cysteine-containing nonapeptide has a heptapeptide sequence homologous to a heptapeptide sequence in a cysteine containing decapeptide from the tryptophanyl-tRNA synthetase of human placenta. The nonapeptide appears to bear the thiol group required for enzyme activity.
D000596:Amino Acids / Q000032:analysis; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D001665:Binding Sites; D004228:Dithionitrobenzoic Acid; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D011487:Protein Conformation; D013438:Sulfhydryl Compounds / Q000032:analysis; D014369:Tryptophan-tRNA Ligase / Q000378:metabolism
D000596:Amino Acids; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D004228:Dithionitrobenzoic Acid; D000604:Amino Acyl-tRNA Synthetases; D014369:Tryptophan-tRNA Ligase