Covalent methionylation of Escherichia coli methionyl-tRNA synthethase: identification of the labeled amino acid residues by matrix-assisted laser desorption-ionization mass spectrometry.
Methionyl-adenylate, the mixed carboxylic-phosphoric acid anhydride synthesized by methionyl-tRNA synthetase (MetRS) is capable of reacting with this synthetase or other proteins, by forming an isopeptide bond with the epsilon-NH2 group of lysyl residues. It is proposed that the mechanism for the in vitro methionylation of MetRS might be accounted for by the in situ covalent reaction of methionyl-adenylate with lysine side chains surrounding the active center of the enzyme, as well as by exchange of the label between donor and acceptor proteins. Following the incorporation of 7.0 +/- 0.5 mol of methionine per mol of a monomeric truncated methionyl-tRNA synthetase species, the enzymic activities of [32P]PPi-ATP isotopic exchange and tRNA(Met) aminoacylation were lowered by 75% and more than 90%, respectively. The addition of tRNA(Met) protected the enzyme against inactivation and methionine incorporation. Matrix-assisted laser desorption-ionization mass spectrometry designated lysines-114, -132, -142 (or -147), -270, -282, -335, -362, -402, -439, -465, and -547 of truncated methionyl-tRNA synthetase as the target residues for covalent binding of methionine. These lysyl residues are distributed at the surface of the enzyme between three regions [114-150], [270-362], and [402-465], all of which were previously shown to be involved in catalysis or to be located in the binding sites of the three substrates, methionine, ATP, and tRNA.
Gillet
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S
Laboratoire de Biochimie (CNRS URA 1970), Ecole Polytechnique, Palaiseau, France. codjo@coli.polytechnique.fr
Hountondji
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C
Schmitter
J M
JM
Blanquet
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S
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000201:enzymology*; D008239:Lysine / Q000378:metabolism; D008715:Methionine / Q000031:analogs & derivatives* / Q000378:metabolism*; D008718:Methionine-tRNA Ligase / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D010446:Peptide Fragments; C011501:methioninyl adenylate; D000249:Adenosine Monophosphate; D008715:Methionine; D008718:Methionine-tRNA Ligase; D008239:Lysine