Enzyme-induced covalent modification of methionyl-tRNA synthetase from Bacillus stearothermophilus by methionyl-adenylate: identification of the labeled amino acid residues by matrix-assisted laser desorption-ionization mass spectrometry.
Methionyl-tRNA synthetase (MetRS) from Bacillus stearothermophilus was shown to undergo covalent methionylation by a donor methionyl-adenylate, the mixed carboxylic-phosphoric acid anhydride synthesized by the enzyme itself. Covalent reaction of methionyl-adenylate with the synthetase or other proteins proceeds through the formation of an isopeptide bond between the carboxylate of the amino acid and the epsilon-NH2 group of lysyl residues. The stoichiometries of labeling, as followed by TCA precipitation, were 2.2 +/- 0.1 and 4.3 +/- 0.1 mol of [14C]Met incorporated by 1 mol of the monomeric MS534 and the native dimeric species of B. stearo methionyl-tRNA synthetase, respectively. Matrix-assisted laser desorption-ionization mass spectrometry designated lysines-261, -295, -301 and -528 (or -534) of truncated methionyl-tRNA synthetase as the target residues for covalent binding of methionine. By analogy with the 3D structure of the monomeric M547 species of E. coli methionyl-tRNA synthetase, lysines-261, -295, and -301 would be located in the catalytic crevice of the thermostable enzyme where methionine activation and transfer take place. It is proposed that, once activated by ATP, most of the methionine molecules react with the closest reactive lysyl residues.
Hountondji
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Laboratoire de Biochemie (CNRS UMR 7654), Ecole Polytechnique, Palaiseau, France.
Beauvallet
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Pernollet
J C
JC
Blanquet
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S
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002250:Carbon Radioisotopes; D002384:Catalysis; D020134:Catalytic Domain; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D008715:Methionine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D008718:Methionine-tRNA Ligase / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001426:Bacterial Proteins; D002250:Carbon Radioisotopes; C011501:methioninyl adenylate; D000249:Adenosine Monophosphate; D008715:Methionine; D008718:Methionine-tRNA Ligase; D008239:Lysine