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

The protonation-reduction of a dioxygen adduct with [LCuI][B(C6F5)4], cupric superoxo complex [LCuII(O2•−)]+ (1), (L=TMG3tren(1,1,1-tris[2-[N2-(1,1,3,3-tetramethylguanidino)]ethyl]amine)), has been investigated. Trifluoroacetic acid (HOAcF) reversibly associates with the superoxo ligand in ([LCuII(O2•−)]+) in a 1:1 adduct [LCuII(O2•−)(HOAcF)]+ (2), as characterized by UV-visible, resonance Raman (rR), nuclear magnetic resonance (NMR) and X-ray absorption (XAS) spectroscopies, along with density functional theory (DFT) calculations. Chemical studies reveal that for the binding of HOAcF with 1 to give 2, Keq = 1.2×105 M−1 (−130 °C) and ΔH° = − 6.9(7) kcal/mol, ΔS° = − 26(4) cal/mol•K). Vibrational (rR) data reveal a significant increase (29 cm−1) in νO-O (= 1149 cm−1) compared to that known for [LCuII(O2•−)]+ (1). Along with results obtained from XAS and DFT calculations, hydrogen bonding of HOAcF to a superoxo O-atom in 2 is established. NMR spectroscopy of 2 at −120 °C in 2-methyltetrahydrofuran are also consistent with 1/HOAcF = 1:1 formulation 2 and that this complex possesses a triplet (S = 1) ground state electronic configuration, as previously determined for 1. The pre-equilibrium acid association to 1 is followed by outer-sphere electron-transfer reduction of 2 by decamethylferrocene (Me10Fc) or octamethylferrocene (Me8Fc), leading to the products H2O2, the corresponding ferrocenium salt and [LCuII(OAcF)]+. Second-order rate constants for electron transfer (ket) were determined to be 1365 M−1 s−1 (Me10Fc) and 225 M−1 s−1 (Me8Fc) at −80 °C. The (bio)chemical relevance of the proton-triggered reduction of the metal-bound dioxygen-derived fragment is discussed.

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“…For PHM and DβM, the ( A )-->( B ) step is postulated as that effecting H-atom abstraction from a R-H substrate, 6b,13 but other views exist. 14 …”

Section: Introductionmentioning

confidence: 99%

Stepwise Protonation and Electron-Transfer Reduction of a Primary Copper–Dioxygen Adduct

et al. 2013

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“…Studies of the kinetic mechanism show that the O 2 and the oxidizable substrate only bind after the reduction of enzyme-bound 2Cu(II) to 2Cu(I) by ascorbate 75 . We found that O 2 binds after hippurate or benzyaldehyde iminooxy acetate 76 rendering the O 2 -requirement for cinnamate inactivation perplexing. Either the order of binding for cinnamate is different, requiring O 2 to bind to reduced PHM before cinnamate can bind, or O 2 binding to the reduced PHM–cinnamate complex results in a subtle rearrangement in the active site to facilitate cinnamate attack by the active site nucleophile.…”

Section: Discussionmentioning

confidence: 93%

Inactivation of peptidylglycineα-hydroxylating monooxygenase by cinnamic acid analogs

McIntyre

Lowe

Battistini

et al. 2015

Journal of Enzyme Inhibition and Medicinal Chemistry

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Peptidylglycine α-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the final reaction in the maturation of α-amidated peptide hormones. Peptidylglycine α-hydroxylating monooxygenase (PHM) is the PAM domain responsible for the copper-, ascorbate- and O2-dependent hydroxylation of a glycine-extended peptide. Peptidylamidoglycolate lyase is the PAM domain responsible for the Zn(II)-dependent dealkylation of the α-hydroxyglycine-containing precursor to the final α-amidated peptide. We report herein that cinnamic acid and cinnamic acid analogs are inhibitors or inactivators of PHM. The inactivation chemistry exhibited by the cinnamates exhibits all the attributes of a suicide-substrate. However, we find no evidence for the formation of an irreversible linkage between cinnamate and PHM in the inactivated enzyme. Our data support the reversible formation of a Michael adduct between an active site nucleophile and cinnamate that leads to inactive enzyme. Our data are of significance given that cinnamates are found in foods, perfumes, cosmetics and pharmaceuticals.

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“…Previous work has established that the addition of the oxidizible substrate occurs after the reduction of enzyme-bound Cu(II) atoms 18e,19. Using ascorbate as the exogenous reductant, Merkler et al 49.…”

Section: Discussionmentioning

confidence: 99%

“…The first step in the catalytic cycle for both enzymes is reduction of the enzyme-bound Cu(II) atoms with an exogenous reductant, most likely ascorbic acid in vivo . Reduction is ping-pong with release of oxidized reductant from E•2Cu(I) followed by binding O 2 or oxidizable substrate to the reduced enzyme 18,19. The dependence of 18 O-kinetic isotope effect on deuteration of the oxidizable substrate for both enzymes indicates that C-H bond cleavage precedes cleavage of the activated O 2 complex, yielding a Cu(II)-hydroperoxo and a substrate-based radical 20.…”

Section: Introductionmentioning

confidence: 99%

Evidence for Substrate Preorganization in the Peptidylglycine α-Amidating Monooxygenase Reaction Describing the Contribution of Ground State Structure to Hydrogen Tunneling

et al. 2010

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Peptidylglycine α-amidating monooxygenase (PAM) is a bifunctional enzyme which catalyzes the post-translational modification of inactive C-terminal glycine-extended peptide precursors to the corresponding bioactive α-amidated peptide hormone. This conversion involves two sequential reactions both of which are catalyzed by the separate catalytic domains of PAM. The first step, the copper-, ascorbate-, and O2-dependent stereospecific hydroxylation at the α-carbon of the C-terminal glycine, is catalyzed by peptidylglycine α-hydroxylating monooxygenase (PHM). The second step, the zinc-dependent dealkylation of the carbinolamide intermediate, is catalyzed by peptidylglycine amidoglycolate lyase. Quantum mechanical tunneling dominates PHM–dependant Cα-H bond activation. This study probes the substrate structure dependence of this chemistry using a set of N-acylglycine substrates of varying hydrophobicity. Primary deuterium kinetic isotope effects (KIEs), molecular mechanical docking, alchemical free energy perturbation, and equilibrium molecular dynamics were used to study the role played by ground-state substrate structure on PHM catalysis. Our data show that all N-acylglycines bind sequentially to PHM in an equilibrium-ordered fashion. The primary deuterium KIE displays a linear decrease with respect acyl chain length for straight-chain N-acylglycine substrates. Docking orientation of these substrates displayed increased dissociation energy proportional to hydrophobic pocket interaction. The decrease in KIE with hydrophobicity was attributed to a pre-organization event which decreased reorganization energy by decreasing the conformational sampling associated with ground state substrate binding. This is the first example of pre-organization in the family of non-coupled copper monooxygenases.

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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

Cited by 14 publications

References 72 publications

Stepwise Protonation and Electron-Transfer Reduction of a Primary Copper–Dioxygen Adduct

Stepwise Protonation and Electron-Transfer Reduction of a Primary Copper–Dioxygen Adduct

Inactivation of peptidylglycineα-hydroxylating monooxygenase by cinnamic acid analogs

Evidence for Substrate Preorganization in the Peptidylglycine α-Amidating Monooxygenase Reaction Describing the Contribution of Ground State Structure to Hydrogen Tunneling

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