Permanent modifications in human acetylcholinesterase by acetylthiocholine or acetylcholine modify the hydrolysis of neutral substrates: phenyl acetate and phenyl valerate as examples

Phenyl acetate (PA) and phenyl valerate (PV) are neutral substrates. PV is used to measure PVase activity of neuropathy target esterase (NTE), which is a key molecular event of organophosphorus-induced delayed neuropathy. However, the interaction of acetylthiocholine (AtCh) with the PVase activity of recombinant human acetylcholinesterase (rhAChE) is not competitive reversible inhibition at the same active site. PVase activity increases when thiocholine (tCh) is released at the active site in the presence of AtCh, after AtCh has been completely hydrolyzed. Kinetic behavior suggested that the potentiation effect is caused by thiocholine released at the active site, where AtCh could act as a Trojan horse. Similarly to AtCh, acetylcholine (ACh) interacts with PVase activity. In this study, we demonstrate that AtCh and ACh can also modify PAase activity. Robust kinetic studies of the interactions between the substrates PA and AtCh were performed. The kinetics did not fit classic competitive model between substrates. We demonstrate that the interaction of AtCh or ACh with the active site is permanent, suggesting covalent or noncovalent modifications to the active site. We conclude that products generated directly at the active site could significantly affect the hydrolysis of substrates in living organisms. These findings have important implications for the practical and biotechnological applications of recombinant and purified cholinesterases.