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Imino-oxy Acetic Acid Dealkylation as Evidence for an Inner-Sphere Alcohol Intermediate in the Reaction Catalyzed by Peptidylglycine α-Hydroxylating Monooxygenase

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posted on 29.07.2009, 00:00 by Neil R. McIntyre, Edward W. Lowe, David J. Merkler
Peptidylglycine α-hydroxylating monooxygenase (PHM, EC catalyzes the stereospecific hydroxylation of a glycyl α-carbon in a reaction that requires O2 and ascorbate. Subsequent dealkylation of the α-hydroxyglycine by another enzyme, peptidylamidoglycolate lyase (PAL. EC, yields a bioactive amide and glyoxylate. PHM is a noncoupled, type II dicopper monooxygenase which activates O2 at only a single copper atom, CuM. 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 [O2] → 0 to 1.0 ± 0.2 as [O2] → ∞ suggesting the dissociation rate constant from the PHM·BIAA complex decreases as [O2] 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 CuII-alkoxide intermediate with a quartet spin state responsible BIAA oxidation.