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

Abstract

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.

Substrate-Induced Carbon Monoxide Reactivity Suggests Multiple Enzyme Conformations at the Catalytic Copper M-Center of Peptidylglycine Monooxygenase

“…14 Despite the lack of spectroscopic evidence upon substrate addition in PHM, kinetic data have established that substrate binding precedes oxygen binding and activation. 4 Strict coupling of oxygen consumption and product formation also suggests that oxygen is activated and ready to commit to catalysis only after substrate binds. 21, 22 FTIR spectroscopy has been the only technique to detect a change upon substrate addition 19 and thus has been utilized here as a probe of substrate-induced structural and electronic perturbations at the catalytic M-center.…”

Copper Active Sites in Biology

“…It should be noted that hippuric acid is the smallest substrate and most versatile for isotope substitution for PHM ( vide infra ), however, many glycine terminal peptides are found to be catalytic for PHM (e.g., Tyr-Val-Gly). 581 These extended peptides bind tighter to PHM (K m ~ 10 µM), which may be due to constructive protein-substrate interactions absent in hippuric acid. Additionally, the substrate dependence on enzyme activity is found to exhibit Michalis-Menton steady-state kinetics for DβM and PHM, while TβM is inhibited (K i = 3.5 mM) by substrate at tyramine concentrations greater than 250 µM.…”

Stopped-Flow Studies of the Reduction of the Copper Centers Suggest a Bifurcated Electron Transfer Pathway in Peptidylglycine Monooxygenase

“…This is the active state of the enzyme, which reacts via equilibrium-ordered binding of first peptide substrate and then dioxygen to form an E•S•O 2 ternary complex (31, 32). The committed step in the enzyme reaction mechanism involves extraction of an electron from the M-site to form a superoxo intermediate (Cu(II)M-O 2 -) (24–26, 33, 34) which is most likely bound end-on and protonated (35).…”