JBC, Vol. 251, Issue 11, 3254-3260, Jun, 1976

Tryptophanyl transfer ribonucleic acid synthetase of Escherichia coli. Character of required thiol group and structure of thiol peptides

G. V. Kuehl, M. L. Lee and K. H. Muench

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.
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