Reaction mechanism of the bicopper enzyme peptidylglycine α-hydroxylating monooxygenase

Abstract

Peptidylglycine α-hydroxylating monooxygenase is a noninteracting bicopper enzyme that stereospecifically hydroxylates the terminal glycine of small peptides for its later amidation. Neuroendocrine messengers, such as oxytocin, rely on the biological activity of this enzyme. Each catalytic turnover requires one oxygen molecule, two protons from the solvent, and two electrons. Despite this enzyme having been widely studied, a consensus on the reaction mechanism has not yet been found. Experiments and theoretical studies favor a pro-S abstraction of a hydrogen atom followed by the rebinding of an OH group. However, several hydrogen-abstracting species have been postulated; because two protons are consumed during the reaction, several protonation states are available. An electron transfer between the copper atoms could play a crucial role for the catalysis as well. This leads to six possible abstracting species. In this study, we compare them on equal footing. We perform quantum mechanics/molecular mechanics calculations, considering the glycine hydrogen abstraction. Our results suggest that the most likely mechanism is a protonation of the abstracting species before the hydrogen abstraction and another protonation as well as a reduction before OH rebinding.

Keywords: Computer Modeling; Copper; Enzyme Catalysis; Kinetic Isotope Effects; Metalloproteins; PHM; Post-translational Modification; QM/MM Simulation; Quantum Tunneling; Reaction Mechanism.

FIGURE 1.

Global reaction catalyzed by PAM as follows: (top) stereospecific hydroxylation of glycine catalyzed by PHM and (bottom) lysis catalyzed by PAL.

FIGURE 2.

Solvent-exposed active site of PHM. The reactant state with [CuOOH]²⁺ as the abstracting species is shown. The small dipeptide Tyr-Gly is used as a model substrate in our calculations. QM atoms are shown as balls. Atom colors are as follows: white (hydrogen), gray (carbon), blue (nitrogen), red (oxygen), yellow (sulfur), and brown (copper).

FIGURE 3.

Schematic view of the structure of the reactant, transition state (TS), and intermediate of the three snapshots. Important bond lengths (in Å) are included in the scheme.

FIGURE 4.

Geometries of the reactant (a, d, and g), transition state (b, e, and h), and intermediate state (c, f, and i) of snapshots 1 (a–c), 2 (d–f), and 3 (g–i) studied in our proposed mechanism. QM atoms are represented by balls and sticks. The remaining part of the substrate and residues His-242, His-244, and Met-314 are represented as sticks. For atom colors, see Fig. 2.

FIGURE 5.

Spin density (contour ± 0.005 atomic units, orange, positive value; green, negative value) of the intermediate state of snapshot 3. The radical at the substrate is delocalized.

FIGURE 6.

Schematic view of the structure of the double protonated intermediate, transition state (TS2), and product of the three snapshots. Important bond lengths (in Å) are included in the scheme.

FIGURE 7.

Geometries of the double protonated intermediates (a, d, and g), transition state (b, e, and h), and product (c, f, and i) of snapshots 1 (a–c), 2 (d–f), and 3 (g–i). No extra electron is considered. See Fig. 4 for the representation.

FIGURE 8.

Mechanism proposed in this paper. See text for details.

FIGURE 9.

Possible abstracting species. Green denotes the most likely mechanism. The ones colored in red are not plausible (the arguments are specified in the text), the one in yellow is plausible, but our mechanism has a lower energy barrier.