Aging mechanism of soman inhibited acetylcholinesterase

Abstract

Acetylcholinesterase (AChE) is a crucial enzyme in the cholinergic nervous system that hydrolyzes neurotransmitter acetylcholine (ACh) and terminates synaptic signals. The catalytic serine of AChE can be phosphonylated by soman, one of the most potent nerve agents, and subsequently undergo an aging reaction. This phosphonylation and aging process leads to irreversible AChE inhibition, results in accumulation of excess ACh at the synaptic clefts, and causes neuromuscular paralysis. By employing Born-Oppenheimer ab initio QM/MM molecular dynamics simulations with umbrella sampling, a state-of-the-art approach to simulate enzyme reactions, we have characterized the aging mechanism of soman phosphonylated AChE and determined its free energy profile. This aging reaction starts with the scission of the O2-Cα bond, which is followed by methyl migration, and results in a tertiary carbenium intermediate. At the transition state, the scissile O2-Cα bond is already cleaved with an average O-C distance of 3.2 ± 0.3 Å and the migrating methyl group is shared between Cα and Cβ carbons with C-C distances of 1.9 ± 0.1 and 1.8 ± 0.1 Å, respectively. The negatively charged phosphonate group is stabilized by a salt bridge with the imidazole ring of the catalytic histidine. A major product of aging, 2,3-dimethyl-2-butanol can be formed swiftly by the reaction of a water molecule. Our characterized mechanism and simulation results provide new detailed insights into this important biochemical process.

Figure 1

Schematic illustration of AChE phosphonylation by soman, re-activation by pralidoxime (2-PAM) and subsequent irreversible aging.

Figure 2

Illustration of overall secondary structure of the non-aged soman inhibited AChE (PDB ID: 2WFZ). The catalytic triad and the covalently bound soman are shown in ball-stick representation and are enlarged to the right panel.

Figure 3

Previously suggested aging reaction mechanisms for soman inhibited AChE.

Figure 4

Illustration of the characterized aging reaction mechanism of soman-phosphonylated AChE.

Figure 5

Free energy profiles for the methyl migration step of aging reaction for the unprotonated Glu199 (red), Glu199Gln mutant (blue) and the protonated Glu199 (green) AChE models. The reaction coordinate was chosen as the difference between two bond lengths: RC = d(somanCβ…somanC5) − d(somanCα…somanC5). The total length of ab initio QM/MM MD simulations for this reaction path is 1.04 ns (Unprotonated Glu199: 20 windows * 20 ps each; Glu199Gln mutant: 17 windows * 20 ps each; protonated Glh199: 16 windows * 20 ps each).

Figure 6

Illustration of key structures along the methyl migration step of aging reaction: (a) unprotonated Glu199, (b) Glu199Gln mutant and (c) protonated Glh199 models. S.S200 is the soman modified serine residue. The labeled distances (A) are averages and standard deviations calculated along the last 15 ps of 20 ps QM/MM MD simulations.

Figure 7

Hydrogen bond network of non-aged soman inhibited AChE active site (a) unprotonated Glu199, (b) Glu199Gln mutant and (c) the protonated Glh199 models. Shown configurations correspond to the 5 ns snapshot from atomistic MD simulation. S.S200 refers the soman modified serine residue. The labeled distances (A) are averages and standard deviations calculated along 20 ns atomistic MD simulations. W1206 in panel (a) is only stable for ~10 ns after which it moves further away from the vicinity of the active site. Hence the average reported in italics for W1206 distance is only for the first 10ns.

Figure 8. Protonated Glh199 model

Free energy profile for hydration of 2,3-dimethyl-butyl carbenium ion. The reaction coordinate was chosen as the difference between two bond lengths: RC = d(watO-watH) − d(somanCβ…watO). The total length of ab initio QM/MM MD simulations for this reaction path is 220 ps (11 windows * 20 ps each).

Figure 9. Protonated Glh199 model

Illustration of key structures along hydration step of the aging reaction. S.S200 is the soman modified serine residue. The distances (A) are averages and standard deviations calculated along the last 15 ps of 20 ps QM/MM MD simulations.