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 massspectrometry

Methionyl-tRNA synthetase (MetRS) from Bacillus stearothermophilus was show n to undergo covalent methionylation by a donor methionyl-adenylate, the mi xed carboxylic-phosphoric acid anhydride synthesized by the enzyme itself. Covalent reaction of methionyl-adenylate with the synthetase or other prote ins proceeds through the formation of an isopeptide bond between the carbox ylate of the amino acid and the epsilon -NH2 group of lysyl residues. The s toichiometries of labeling, as followed by TCA precipitation, were 2.2 +/- 0.1 and 4.3 +/- 0.1 mol of [C-14]Met incorporated by 1 mol of the monomeric MS534 and the native dimeric species of B. stearo methionyl-tRNA synthetas e, respectively. Matrix-assisted laser desorption-ionization mass spectrome try designated lysines-261, -295, -301 and -528 (or -534) of truncated meth ionyl-tRNA synthetase as the target residues for covalent binding of methio nine. By analogy with the 3D structure of the monomeric M547 species of E. coli methionyl-tRNA synthetase, lysines-261, -295, and -301 would be locate d in the catalytic crevice of the thermostable enzyme where methionine acti vation and transfer take place. It is proposed that, once activated by ATP, most of the methionine molecules react with the closest reactive lysyl res idues.