collapses to release the amine and the enzyme-bound acyl intermediate. Lys142 acts
as a general base-general acid, mediating both the deprotonation of Ser241 and
subsequent protonation of the leaving group, shuttled through Ser217. The reaction
terminates with a water-mediated deacylation of the acyl enzyme-bound intermediate [4, 7, 8, 51]. The mechanistic characterization of the hydrolysis of oleamide
and similar compounds has been computationally carried out with QM/MM calculations [52–56]. Furthermore, studies on drug-design computational perspectives
for inhibitors of FAAH have appeared to [57–59].
The present study provides an extensive atomic-level molecular dynamics
structural analysis for the FAAH-anandamide complex, of both monomer and dimerized structures of the enzyme. The intent was also to discover key players at the
active site complexed with anandamide. An extensive analysis was performed on
the protein stability during MD, residue flexibility, and analysis of the SASA, for
both monomer and dimer complexes of FAAH-anandamide. Additionally, the water
distribution around anandamide and a hydrogen bond analysis for the substrate
inserted in the active site were considered.
In sum, the results provide detailed atomistic insights that include the dynamic
effect of the systems and the effect of the solvent, complementing the static view
that could be obtained from the X-ray structures available for this enzyme, and
contributing with important indications for future mechanistic and drug-design
studies on this important enzyme, allowing a better understanding of FAAH.
2 Methodology
The AMBER 10 [60] molecular dynamics package was used in all the molecular
dynamics simulations performed. The monomer and dimer systems were prepared
from the 1MT5 X-ray crystallographic structure of FAAH complexed with an
arachidonyl inhibitor (methoxy arachidonyl fluorophosphonate) [3]. The full X-ray
structure contains a total of 32 subunits, each one with 572 amino acids. A monomer
and a dimer were prepared from this structure and considered for the MD study. In
both systems, the substrate analogue methoxy arachidonyl fluorophosphonate was
modelled into the endogenous cannabinoid substrate anandamide. The Duan et al.
2002 [61] parameters were employed to describe the protein, while the
ANTECHAMBER module [62] of AMBER and the General AMBER Force Field
(GAFF) [63] were used to parameterize the anandamide substrate (Fig. 1), with
charges derived with RESP at the HF/6-31G(d) level of theory, to be coherent with
all other parameters used.
Conventional protonation states for all amino acids at pH 7 were considered. All
the hydrogen atoms were added and counter-ions (Cl
− ) were employed to neutralize
the positive charge of the system (ranging from −3 to −6, in the monomer and
dimer studies, respectively). The Leap program was used in this regard. Each
system was placed in its own rectangular box containing a minimum distance of
12 Å of TIP3P water molecules between the enzyme and the box side. The size of
Molecular Dynamics Analysis of FAAH Complexed with Anandamide
117
as a general base-general acid, mediating both the deprotonation of Ser241 and
subsequent protonation of the leaving group, shuttled through Ser217. The reaction
terminates with a water-mediated deacylation of the acyl enzyme-bound intermediate [4, 7, 8, 51]. The mechanistic characterization of the hydrolysis of oleamide
and similar compounds has been computationally carried out with QM/MM calculations [52–56]. Furthermore, studies on drug-design computational perspectives
for inhibitors of FAAH have appeared to [57–59].
The present study provides an extensive atomic-level molecular dynamics
structural analysis for the FAAH-anandamide complex, of both monomer and dimerized structures of the enzyme. The intent was also to discover key players at the
active site complexed with anandamide. An extensive analysis was performed on
the protein stability during MD, residue flexibility, and analysis of the SASA, for
both monomer and dimer complexes of FAAH-anandamide. Additionally, the water
distribution around anandamide and a hydrogen bond analysis for the substrate
inserted in the active site were considered.
In sum, the results provide detailed atomistic insights that include the dynamic
effect of the systems and the effect of the solvent, complementing the static view
that could be obtained from the X-ray structures available for this enzyme, and
contributing with important indications for future mechanistic and drug-design
studies on this important enzyme, allowing a better understanding of FAAH.
2 Methodology
The AMBER 10 [60] molecular dynamics package was used in all the molecular
dynamics simulations performed. The monomer and dimer systems were prepared
from the 1MT5 X-ray crystallographic structure of FAAH complexed with an
arachidonyl inhibitor (methoxy arachidonyl fluorophosphonate) [3]. The full X-ray
structure contains a total of 32 subunits, each one with 572 amino acids. A monomer
and a dimer were prepared from this structure and considered for the MD study. In
both systems, the substrate analogue methoxy arachidonyl fluorophosphonate was
modelled into the endogenous cannabinoid substrate anandamide. The Duan et al.
2002 [61] parameters were employed to describe the protein, while the
ANTECHAMBER module [62] of AMBER and the General AMBER Force Field
(GAFF) [63] were used to parameterize the anandamide substrate (Fig. 1), with
charges derived with RESP at the HF/6-31G(d) level of theory, to be coherent with
all other parameters used.
Conventional protonation states for all amino acids at pH 7 were considered. All
the hydrogen atoms were added and counter-ions (Cl
− ) were employed to neutralize
the positive charge of the system (ranging from −3 to −6, in the monomer and
dimer studies, respectively). The Leap program was used in this regard. Each
system was placed in its own rectangular box containing a minimum distance of
12 Å of TIP3P water molecules between the enzyme and the box side. The size of
Molecular Dynamics Analysis of FAAH Complexed with Anandamide
117