2 research outputs found
QM/MM Molecular Dynamics Investigations of the Substrate Binding of Leucotriene A4 Hydrolase: Implication for the Catalytic Mechanism
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
Leukotriene A4 hydrolase (LTA4H)
functions as a mono-zinc bifunctional
enzyme with aminopeptidase and epoxidase activities. While the aminopeptidase
mechanism is well understood, the epoxidase mechanism remains less
clear. In continuation of our prior research, we undertook an in-depth
exploration of the LTA4H catalytic role as an epoxidase, employing
a combined SCC-DFTB/CHARMM method. In the current work, we found that
the conversion of LTA4 to leukotriene B4 (LTB4) involves three successive
steps: epoxy ring opening (RO), nucleophilic attack (NA), and proton
transfer (PT) reactions at the epoxy oxygen atom. Among these steps,
the RO and NA stages constitute the potential rate-limiting step within
the entire epoxidase mechanism. Notably, the NA step implicates D375
as the general base catalyst, while the PT step engages protonated
E271 as the general acid catalyst. Additionally, we delved into the
mechanism behind the formation of the isomer product, Δ6-trans-Δ8-cis-LTB4. Our findings debunked the feasibility of a direct LTB4 to iso-LTB4 conversion. Instead, we highlight the possibility
of isomerization from LTA4 to its isomeric conjugate (iso-LTA4), showing comparable energy barriers of 5.1 and 5.5 kcal/mol
in aqueous and enzymatic environments, respectively. The ensuing dynamics
of iso-LTA4 hydrolysis subsequently yield iso-LTB4
via a mechanism akin to LTA4 hydrolysis, albeit with a heightened
barrier. Our computations firmly support the notion that substrate
isomerization exclusively takes place prior to or during the initial
substrate-binding phase, while LTA4 remains the dominant conformer.
Notably, our simulations suggest that irrespective of the active site’s
constrained L-shape, isomerization from LTA4 to its isomeric conjugate
remains plausible. The mechanistic insights garnered from our simulations
furnish a valuable understanding of LTA4H’s role as an epoxidase,
thereby facilitating potential advancements in inhibitor design