INCORPORATION OF 2 O-18 ATOMS INTO A PEPTIDE DURING ISOASPARTYL REPAIR REVEALS REPEATED PASSAGE THROUGH A SUCCINIMIDE INTERMEDIATE

To study the mechanism of protein carboxyl methyltransferase-driven re pair of age-damaged sites in polypeptides, a model L-isoaspartyl pepti de, L-isotetragastrin, was enzymatically repaired to normal L-tetragas trin in the presence of O-18-enriched water. By this design, the enric hment of O-18 atoms in the peptide would reflect the number of passage s through a hydrolyzable succinimide intermediate during formation of the repaired product. Mass determinations by FAB mass spectrometry rev ealed repaired peptide with two O-18 atoms incorporated, demonstrating that more than a single cycle of methylation and demethylation is nec essary to ensure stoichiometric repair.