Imino-oxy acetic acid dealkylation as evidence for an inner-sphere alcohol intermediate in the reaction catalyzed by peptidylglycine alpha-hydroxylating monooxygenase.
Peptidylglycine alpha-hydroxylating monooxygenase (PHM, EC 1.14.17.3) catalyzes the stereospecific hydroxylation of a glycyl alpha-carbon in a reaction that requires O(2) and ascorbate. Subsequent dealkylation of the alpha-hydroxyglycine by another enzyme, peptidylamidoglycolate lyase (PAL. EC 4.3.2.5), yields a bioactive amide and glyoxylate. PHM is a noncoupled, type II dicopper monooxygenase which activates O(2) at only a single copper atom, Cu(M). In this study, the PHM mechanism was probed using a non-natural substrate, benzaldehyde imino-oxy acetic acid (BIAA). PHM catalyzes the O-oxidative dealkylation of BIAA to benzaldoxime and glyoxylate with no involvement of PAL. The minimal kinetic mechanism for BIAA was shown to be steady-state ordered using primary deuterium kinetic isotope effects. The (D)(V/K)(APPARENT, BIAA) decreased from 14.7 +/- 1.0 as [O(2)] --> 0 to 1.0 +/- 0.2 as [O(2)] --> infinity suggesting the dissociation rate constant from the PHM x BIAA complex decreases as [O(2)] increases; thereby, reducing the steady-state concentration of [PHM](free). BIAA was further used to differentiate between potential oxidative Cu/O species using a QM/MM reaction coordinate simulation to determine which species could yield product O-dealkylation that matched our experimental data. The results of this study provided compelling evidence for the presence of a covalently linked Cu(II)-alkoxide intermediate with a quartet spin state responsible BIAA oxidation.
McIntyre
Neil R
NR
Department of Chemistry, University of South Florida, 4202 East Fowler Avenue, Tampa, Florida 33620, USA.
Lowe
Edward W
EW
Merkler
David J
DJ
D000085:Acetates / Q000138:chemical synthesis* / Q000737:chemistry; D000438:Alcohols / Q000737:chemistry*; D000478:Alkylation; D001205:Ascorbic Acid / Q000737:chemistry; D055162:Biocatalysis; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D010100:Oxygen / Q000737:chemistry; D013237:Stereoisomerism
D000085:Acetates; D000438:Alcohols; D009097:Multienzyme Complexes; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D001205:Ascorbic Acid; D010100:Oxygen