title: The source of the oxygen atom in the alpha-hydroxyglycine intermediate of the peptidylglycine alpha-amidating reaction.
abstract: 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 H2(18)O-enriched medium or under an atmosphere of 18O2, and isolated the alpha-hydroxylglycine 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 monooxygenase reaction.
authors.affiliation: Department of Biochemistry, Fukushima Medical College, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Noguchi
authors.forename: H
authors.initials: H
authors.lastname: Seino
authors.forename: H
authors.initials: H
authors.lastname: Kochi
authors.forename: H
authors.initials: H
authors.lastname: Okamoto
authors.forename: T
authors.initials: T
authors.lastname: Tanaka
authors.forename: M
authors.initials: M
authors.lastname: Hirama
mesh_terms: D019760:Amidine-Lyases; D000595:Amino Acid Sequence; D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism; D001921:Brain / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D003300:Copper / Q000494:pharmacology; D005998:Glycine / Q000378:metabolism*; D006900:Hydroxylation; D008190:Lyases / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009097:Multienzyme Complexes; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D051381:Rats; D016339:Spectrometry, Mass, Fast Atom Bombardment
chemical_list: D009097:Multienzyme Complexes; D009842:Oligopeptides; D003300:Copper; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D008190:Lyases; D019760:Amidine-Lyases; C068521:peptidylamidoglycolate lyase; D001205:Ascorbic Acid; D010100:Oxygen; D005998:Glycine