Substrate Binding to the Acetylcholinesterase Peripheral Site Promotes Substrate Hydrolysis but also Gives Rise to Substrate Inhibition

Abstract

Many ligands that bind to the peripheral site of acetylcholinesterase (AChE) inhibit substrate hydrolysis at the acylation site, though the interpretation of this inhibition has been unclear. Since previous explanations based upon equilibrium ligand binding cannot be justified for AChE, we introduced an alternative nonequilibrium analysis. This analysis incorporates a steric blockade mechanism which assumes that the only effects of a small bound peripheral ligand are to slow down the rates of substrate association and dissociation at the acylation site and concomitantly reduce the product dissociation rate. Experimental data using propidium and gallamine as peripheral site ligands and acetylthiocholine, phenyl acetate, huperzine A and m-((N, N, N -trimethylammonio)-trifluoroacetophenone as acylation site ligands agreed well with our theory. The nonequilibrium model has been extended to account for substrate inhibition based upon acetylthiocholine affinity for the peripheral site. This affinity was determined by measuring acetylthiocholine inhibition of the association rate k on for the slowly equilibrating peripheral site ligand fasciculin 2. The measured K s of about 1–2 mM was considerably lower than the K ss estimate of about 20 mM determined by application of the Haldane equation to the steady-state substrate inhibition curve. A better understanding of the interaction between peripheral site ligands and acetylcholinesterase may help to understand the physiological role of the peripheral site and to design new ligands that inhibit organophosphorylation.