Incorporation of two 18O atoms into a peptide during isoaspartyl repair reveals repeated passage through a succinimide intermediate.

To study the mechanism of protein carboxyl methyltransferase-driven repair of age-damaged sites in polypeptides, a model L-isoaspartyl peptide, L-isotetragastrin, was enzymatically repaired to normal L-tetragastrin in the presence of 18O-enriched water. By this design, the enrichment of 18O atoms in the peptide would reflect the number of passages through a hydrolyzable succinimide intermediate during formation of the repaired product. Mass determinations by FAB mass spectrometry revealed repaired peptide with two 18O atoms incorporated, demonstrating that more than a single cycle of methylation and demethylation is necessary to ensure stoichiometric repair.