Proton shuttles and phosphatase activity in soluble epoxide hydrolase

Abstract

Recently, a novel metal Mg2+-dependent phosphatase activity has been discovered in the N-terminal domain of the soluble epoxide hydrolase (sEH), opening a new branch of fatty acid metabolism and providing an additional site for drug targeting. Importantly, the sEH N-terminal fold belongs to the haloacid dehalogenase (HAD) superfamily, which comprises a vast majority of phosphotransferases. Herein, we present the results of a computational study of the sEH phosphatase activity, which includes classical molecular dynamics (MD) simulations and mixed quantum mechanical/molecular mechanics (QM/MM) calculations. On the basis of experimental results, a two-step mechanism has been proposed and herein investigated: (1) phosphoenzyme intermediate formation and (2) phosphoenzyme intermediate hydrolysis. Building on our earlier work, we now provide a detailed description of the reaction mechanism for the whole catalytic cycle along with its free energy profile. The present computations suggest metaphosphate-like transition states for these phosphoryl transfers. They also reveal that the enzyme promotes water deprotonation and facilitates shuttling of protons via a metal-ligand connecting water bridge (WB). These WB-mediated proton shuttles are crucial for the activation of the solvent nucleophile and for the stabilization of the leaving group. Moreover, due to the conservation of structural features in the N-terminal catalytic site of sEH and other members of the HAD superfamily, we suggest a generalization of our findings to these other metal-dependent phosphatases.

Figure 1

Cartoon of the sEH N-terminal domain fold. Secondary structures are colored in yellow (B-sheets), violet (alpha-helixes) and green (loops); the linker is colored in red. The orange sphere indicates the Mg²⁺ cofactor present in the active site, while coordinating ligands and the substrate molecule are depicted in stick representation and colored based on atom-type.

Figure 2

Selected snapshots taken from our computer simulations of the two investigated phosphoryl transfers comprising the catalytic cycle. Top: nucleophilic attack of Asp9 at the Mg2+ coordinated phosphoryl group, with substrate cleavage and phosphoenzyme intermediate formation INTa. In the middle, the transition state structure TS1 showing the concomitant proton shuttle (labeled PT1 and PT2) from a Mg2+ coordinated water molecule to the leaving group oxygen via a bridging solvent water. Bottom: second phosphoryl transfer from the phospho-Asp9 to one attacking solvent water, leading to the product state, with now a second proton shuttle (labeled PT3 and PT4 in TS2) traveling in the reverse direction to create the nucleophile OH^-.

Figure 3

Free energy profile (top) and selected average bond distances (bottom) along the first catalytic step of phosphoenzyme formation (INTa). Bond distance labels as in Figure 2; notably r1 and r2 the breaking and forming P-O bond lengths respectively. The proton shuttle occurs at RC ≈ -0.5A (vertical dashed line), just before the system reaches the TS plateau (orange region). Note the shortening of the Mg2+-ligand distance, d1, upon proton donation and transfer along the H-bond wire (d2/d3 and d4/d5 crossing).

Figure 4

Free energy profile (top) and selected average bond distances (bottom) along the second catalytic step. Bond distance labels as in Figure 2 (INTb panel). Here the proton shuttle (dashed vertical line) occurs in the reverse direction (note the d2/d3 and d4/d5 crossing) after the TS plateau (orange region).

Figure 5

Average charges of selected molecular moieties in the active site along the first (left panel) and second (right panel) phosphoryl transfer reaction (see color coding in Figure). The metaphosphate-like transition state formation along both transfers is shown by the temporary increase of negative charge of the PO₃^- group (red line) along the reaction. The crossing of blue and violet lines shows the charge transfer along the H-bond wire during the proton shuttles (dashed vertical lines).

Scheme 1

Mechanism of phosphatase activity in sEH proposed by Gomez G. A. et al (23), and investigated in our study: Step1) Phosphoenzyme intermediate formation via a nucleophilic attack at the phosphate group of the phosphoester substrate by Asp9; Step2) Phosphoenzyme hydrolysis via a nucleophilic attack of a water molecule at the scissile phosphorus atom.