Covalent methionylation of escherichia coli methionyl‐tRNA synthethase: Identification of the labeled amino acid residues by matrix‐assisted laser desorption‐ionization mass spectrometry

Abstract

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 ϵ-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]PP_i-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.