Labelling of L-isoleucine tRNA ligase from Escherichia coli with L-isoleucyl-bromomethyl ketone.
L-Isoleucine: tRNA ligase from Escherichia coli could be irreversibly inactivated by L-isoleucyl-bromomethyl ketone but not by L-isoleucyl-chromethyl ketone. The inactivation rate exhibited a saturation concentration dependence typical for an affinity reagent. L-Isoleucine provided 100% protection against inactivation at saturating concentration, whereas ATP, AMP, and pyrophosphate offered partial protection and tRNAIle, no protection. The ligase was labelled in preparative scale with L-[14C]isoleucyl-bromethyl ketone. The molar ration of label incorporated to enzyme inactivated was close to unity. The protein was subsequently subjected to tryptic digestion and the radioactive peptide isolated and identified. The labelled amino acid proved to be the same cysteine previously reported as being labelled with N-[14C]ethylmaleimide [Kula, M.-R. (1974) FEBS Lett. 46, 130-133].
D000345:Affinity Labels; D000596:Amino Acids / Q000032:analysis; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001665:Binding Sites; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D007532:Isoleucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D007533:Isoleucine-tRNA Ligase / Q000378:metabolism*; D007659:Ketones / Q000494:pharmacology; D007700:Kinetics; D011485:Protein Binding
D000345:Affinity Labels; D000596:Amino Acids; D007659:Ketones; D007532:Isoleucine; D004228:Dithionitrobenzoic Acid; D000604:Amino Acyl-tRNA Synthetases; D007533:Isoleucine-tRNA Ligase; D004229:Dithiothreitol