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 eNH2 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 ("P)PPi-ATP isotopic exchange and tRNAM" aminoacylation were lowered by 75% and more than 90%, respectively. The addition of tRNAMe* protected the enzyme against inactivation and methionine incorporation. Matrix-assisted laser desorption-ionization mass Spectrometry designated lysines-I 14, -1 32, -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), (270-3621, and (402-4651, 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.