Introduction
Acetylcholinesterase (AChE) is the enzyme responsible for the
termination of impulse transmission in the central nervous system. It
rapidly hydrolyzes acetylcholine
(ACh), the neurotransmitter in the synaptic cleft at the neuromuscular
junction and at cholinergic synapses.1 The catalytic
efficiency is very high, approaching the rate of a diffusion-controlled
reaction.2,3AChE is the target of various
natural and synthetic compounds such as organophosphorus (OP) nerve
agents and pesticides.4 By inhibiting AChE, OP
compounds cause accumulation of ACh, resulting in paralysis, seizures,
and other symptoms of the cholinergic syndrome, and even lead to death
by respiratory arrest. Since the level of ACh is associated with
various neurological disorders,
the inhibition of AChE is also a
therapeutic strategy for the treatment of related diseases. The AChE
inhibitors have provided the principal drugs approved by the FDA for the
management of Alzheimer’s disease.4,5
The binding pocket of AChE is a long and narrow gorge, extending from
the surface of the enzyme down to the catalytic site. In this site, the
catalytic triad (Ser203, His447, Glu334) and the oxyanion hole (Gly121,
Gly122, Ala204) are essential in the catalytic reaction.
The Glu202, a residue adjacent to
the catalytic His447, also plays important role in
catalysis.6-8 With substitutions at Glu202, the
catalytic rate constant was decreased up to 80-fold compared with
wild-type AChE.9 It has also been suggested that
Glu202 is a key residue facilitating the HI6-induced reactivation of the
sarin-inhibited AChE.10 In addition, Glu202 has been
reported as a key residue of AChE with an important role in
phosphorylation, 7 spontaneous
reactivation11, and aging 8.
Although Glu202 has long been
considered as negatively charged in many studies, there exists evidence
suggesting that Glu202 is more likely to be protonated. For example,
AChE is more stable when both Glu202 and Glu450 are
protonated.12 Also, it has been suggested that Glu202
needs to be protonated for reactivation to occur.13However, Glu202 is on the surface
of the catalytic site and is freely accessible by water molecules. It
seems reasonable for Glu202 to majorly take the deprotonated state. Why
Glu202 more likely to adopt the protonated state is still not fully
understood.
Butyrylcholinesterase (BChE) is a plasma cholinesterase. It is
structurally very similar to AChE. In BChE, the Glu197 is a residue
corresponding to the Glu202 in AChE. In our previous study, we found
that the protonation state of Glu197 in BChE is crucial in maintaining
the catalytic triad.14 Shown in Figure 1A is the
crystal structure of BChE. Our study demonstrated that the Glu197 needs
to be protonated to join and stabilize a key hydrogen bond network,
which has a highly conserved water molecule at its center. Without the
support of this key hydrogen bond network, the catalytic His438
dramatically shifts away from its crystal structure position, resulting
in a distorted catalytic triad that is unable to perform catalysis. If
Glu197 is deprotonated, then repulsion occurs between the negatively
charged water oxygen and the negatively charged carboxyl group of
Glu197, pushing Glu197 away from the key hydrogen bond network. Without
the stabilization effect from Glu197, the key hydrogen bond network
eventually collapses, affecting the stability of catalytic H447 and
consequently caused a distorted catalytic triad.