QM/MM Molecular Dynamics Investigations of the Substrate Binding of Leucotriene A4 Hydrolase: Implication for the Catalytic Mechanism

Abstract

LTA4H is a monozinc bifunctional enzyme which exhibits both aminopeptidase and epoxide hydrolase activities. Its dual functions in anti- and pro-inflammatory roles have attracted wide attention to the inhibitor design. In this work, we tried to construct Michaelis complexes of LTA4H with both a native peptide substrate and LTA4 molecule using combined quantum mechanics and molecular mechanics molecular dynamics simulations. First of all, the zinc ion is coordinated by H295, H299, and E318. For its aminopeptidase activity, similar to conventional peptidases, the fourth ligand to the zinc ion is suggested to be an active site water, which is further hydrogen bonded with a downstream glutamic acid, E296. For the epoxide hydrolase activity, the fourth ligand to the zinc ion is found to be an epoxy oxygen atom. The potential of mean force calculation indicates about an 8.5 kcal/mol activation barrier height for the ring-opening reaction, which will generate a metastable carbenium intermediate. Subsequent frontier molecular orbital analyses suggest that the next step would be the nucleophilic attacking reaction at the C12 atom by a water molecule activated by D375. Our simulations also analyzed functions of several important residues like R563, K565, E271, Y383, and Y378 in the binding of peptide and LTA4.

Quantum Mechanical/Molecular Mechanical Elucidation of the Catalytic Mechanism of Leukotriene A4 Hydrolase as an Epoxidase

“…Since we simulated the initial step of the epoxy ring opening in our previous work at the SCC-DFTB/CHARMM level of theory, we simply accept those results here. Consequently, this study will solely focus on NA and PT reactions.…”

“…We constructed an LTA4H/LTA4 complex model in our previous work, based on the crystallographic structure (PDB ID 2VJ8), which depicts wild-type LTA4H cocrystallized with the inhibitor hydroxamic acid. To assess the model stability and substrate-binding features, we conducted extensive SCC-DFTB/MM molecular dynamics (MD) simulations.…”

“…Protonated E271 serves as a stabilizing factor for the active-site water molecule. According to a recent X-ray structure and our previous simulations, the initial structure depicted in Scheme S2B appears to be preferable for the hydrolysis of LTA4. As a result, all of the calculations presented in this study adhere to this model.…”

“…Here, following our previous work, in which we proposed a model for the LTA4H/LTA4 complex and elucidated the first step of epoxy ring opening, our focus shifted toward the subsequent steps. We aimed to comprehensively address the entire hydrolysis reaction of LTA4 and delve into the mechanism of enantioselectivity.…”

Theoretical Characterization of the Step-by-Step Mechanism of Conversion of Leukotriene A4 to Leukotriene B4 Catalysed by the Enzyme Leukotriene A4 Hydrolase

“…To our knowledge, only a theoretical study of the epoxide hydrolase reaction exists so far [ 23 ]. However, this work was just restricted to the step corresponding to the epoxy ring opening, using the self-consistent-charge density-functional tight-binding (SCC-DFTB) theory [ 24 , 25 , 26 ] for the Quantum Mechanics calculations and the CHARMM force field for the molecular mechanics part [ 27 ].…”