THE SOURCE OF THE OXYGEN ATOM IN THE ALPHA-HYDROXYGLYCINE INTERMEDIATE OF THE PEPTIDYLGLYCINE ALPHA-AMIDATING REACTION

Peptidylglycine alpha-amidating activity catalyses the oxidation of a C-terminally glycine-extended peptide to a desglycine alpha-amidated peptide at the expense of ascorbate and O2 in the presence of Cu2+. The reaction involves oxidative N-dealkylation within the terminal glycine residue, with retention of the glycine N atom and release of the remainder as glyoxylate. Recent studies by us and others have revealed that the reaction consists of two steps via a carbinolamide as an intermediate (peptidyl alpha-hydroxyglycine), and also that two separate enzymes derived from a common precursor protein catalyse these steps, formation of the carbinolamide and its conversion into alpha-amide and glyoxylate. As for the mechanism of carbinolamide formation, two distinct pathways can be considered: direct mono-oxygenation at the glycine-alpha-C atom and dehydrogenation leading to an imine followed by hydration. To draw a distinction between them, we carried out the reaction with D-Tyr-Val-Gly as the substrate either in the (H2O)-O-18-enriched medium or under an atmosphere of O-18(2), and isolated the alpha-hydroxyglycine intermediate. The fast-atom-bombardment mass-spectral analysis demonstrated that the hydroxy O atom comes from O2, but not from H2O, indicating that the alpha-hydroxylation should be a mono-oxygenase reaction.