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.