Nerve agents, a subfamily of organophosphorus compounds (OPs), have a history of utilization as chemical weapons and insecticides. There are two main families of chemical weapons, described as the G-series, and V-series nerve agents. The main structural difference between these two families is that G-series nerve agents contain a small fluoride leaving group, while the V-series nerve agents possess a larger amino-thiolate leaving group. These compounds exert their toxicity via the inhibition of acetylcholinesterase (AChE), leading to a buildup of acetylcholine and resulting in overstimulation of cholinergic receptors. The active site of acetylcholinesterase is the typical serine hydrolase catalytic triad, comprised of Ser203, His447, and Glu334 (numbering for human AChE).1
OPs inhibit AChE through phosphylation of the catalytic serine and the formation of a stable OP-AChE adduct (), thereby resulting in a P–O bond that is resistant to cleavage by water molecules in the gorge. Although enzymatic function can be regenerated by cleaving the P–O(Ser) bond using a stronger nucleophile, most commonly an oxime,2
reactivation of the native serine is complicated by inefficient reactivation by oximes for specific OPs; frequently, the efficacy of an oxime is correlated to a specific OP nerve agent. In addition, the OP adduct can undergo dealkylation (aging) which renders the enzyme unreactivatable by common methods.3
Proposed Mechanism for Inhibition of AChE by OPs.
Due to the difficulty in regenerating cholinesterase activity, a detailed study of the enzymolysis of OPs was undertaken previously with the hopes of creating a mutant enzyme capable of catalyzing the hydrolysis of nerve agents.4,5,6
One of the more interesting mutations, a substitution of the catalytic serine for cysteine (S203C) was found to render the protein inactive against CMP,7
an OP with a coumarin leaving group. A chemically similar mutation (Ser/Cys to selenocysteine) in other enzymes has resulted in increases of 100–500× activity over the corresponding wild-type enzyme.8
It is, therefore, not clear why the substitution of a stronger nucleophile in the form of cysteine resulted in an inactive enzyme for the S203C mutant.
The hydrolysis of OPs has a long history of study, both experimentally and computationally, that suggests the hydrolysis of nerve agents proceeds via an addition-elimination pathway, forming a stable penta-coordinated phosphorus intermediate.9,10,11,12,13
Along this pathway, there are two main transition state structures: the first corresponds to the association between nucleophile and OP, which forms a pentacoordinate intermediate, and the second transition state corresponds to cleavage of the OP-leaving group bond, followed by dissociation of the complex. Depending on the OP, there may also be additional transition states that involve reorientations of the O-
alkyl substituent. Predicted solvent effects on the potential energy surface vary between studies, with some studies suggesting solvation increases the association barrier while decreasing the decomposition barrier,9
and another predicting solvation will decrease both barriers.10
The majority of these studies have not optimized the OP in an implicit polarizable continuum model (PCM) solvent representation; instead, they have computed single-point energy evaluations with implicit solvation using the gas-phase geometries, which may be the cause of this disagreement. However, the majority of computational studies indicate that the association between nucleophile and OP is the rate-limiting step, and have accurately reproduced the observed experimental ~9 kcal/mol energy barrier of GB.14
Several studies have expanded beyond aqueous hydrolysis in order to study the reaction at the catalytic site, using small fragments of the active site,15
as well as the full enzymatic environment through quantum mechanical, molecular mechanical (QM/MM) calculations.16,17,18
These studies predict the presence of a stable penta-coordinate intermediate, similar to the isolated systems. However, studies that utilize a full enzymatic approach suggest that, in the active site, decomposition of the intermediate is the rate-limiting step, at least for the acylation/deacylation of AChE.16
This study aims to determine the potential energy surface for phosphonylation of the AChE active site by GB, as well as to clarify the roles that solvation and active-site residues play in this reaction. Results will be compared to the potential energy surface for aqueous hydrolysis of GB by both ethoxide (CH3
) and ethyl thiolate (CH3
). In addition to wild-type (wt) AChE, the previously studied S203C mutant7
is investigated in order to elucidate the cause of the experimentally observed inactivity.