COVALENT METHIONYLATION OF ESCHERICHIA-COLI METHIONYL-TRANSFER-RNA SYNTHETASE - IDENTIFICATION OF THE LABELED AMINO-ACID-RESIDUES BY MATRIX-ASSISTED LASER DESORPTION-IONIZATION MASS-SPECTROMETRY

Methionyl-adenylate, the mixed carboxylic-phosphoric acid anhydride sy nthesized 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 lysin e side chains surrounding the active center of the enzyme, as well as by exchange of the label between donor and acceptor proteins. Followin g the incorporation of 7.0 +/- 0.5 mol of methionine per mol of a mono meric truncated methionyl-tRNA synthetase species, the enzymic activit ies of [P-32]PP1-ATP isotopic exchange and tRNA(Met) aminoacylation we re lowered by 75% and more than 90%, respectively. The addition of tRN A(Met) protected the enzyme against inactivation and methionine incorp oration. 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 re sidues are distributed at the surface of the enzyme between three regi ons [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 site s of the three substrates, methionine, ATP, and tRNA.