NONEQUILIBRIUM ANALYSIS ALTERS THE MECHANISTIC INTERPRETATION OF INHIBITION OF ACETYLCHOLINESTERASE BY PERIPHERAL SITE LIGANDS

The active site gorge of acetylcholinesterase (AChE) contains two site s of ligand binding, an acylation site near the base of the gorge with a catalytic triad characteristic of serine hydrolases, and a peripher al site at the mouth of the gorge some 10-20 Angstrom from the acylati on site, Many ligands that bind exclusively to the peripheral site inh ibit substrate hydrolysis at the acylation site, but the mechanistic i nterpretation of this inhibition has been unclear. Previous interpreta tions have been based on analyses of inhibition patterns obtained from steady-state kinetic models that assume equilibrium ligand binding. T hese analyses indicate that inhibitors bound to the peripheral site de crease acylation and deacylation rate constants and/or decrease substr ate affinity at the acylation site by factors of up to 100. Conformati onal interactions have been proposed to account for such large inhibit ory effects transmitted over the distance between the two sites, but s ite-specific mutagenesis has failed to reveal residues that mediate th e proposed conformational linkage, Since examination of individual rat e constants in the AChE catalytic pathway reveals that assumptions of equilibrium ligand binding cannot be justified, we introduce here an a lternative nonequilibrium analysis of the steady-state inhibition patt erns. This analysis incorporates a ste! ic blockade hypothesis which a ssumes that the only effect of a bound peripheral site ligand is to de crease the association and dissociation rate constants for an acylatio n site ligand without altering the equilibrium constant for ligand bin ding to the acylation site, Simulations based on this nonequilibrium s teric blockade model were in good agreement with experimental data for inhibition by the peripheral site ligands propidium and gallamine at low concentrations of either acetylthiocholine or phenyl acetate if bi nding of these ligands slows substrate association and dissociation ra te constants by factors of 5-70, Direct measurements with the acylatio n site ligands huperzine A and m-(N,N,N-trimethylammonio)trifluoroacet ophenone showed that bound propidium decreased the association rate co nstants 49- and 380-fold and the dissociation rate constants 10- and 6 0-fold, respectively, relative to the rate constants for these acylati on site ligands with free AChE, in reasonable agreement with the noneq uilibrium steric blockade model, We conclude that this model can accou nt for the inhibition of AChE by small peripheral site ligands such as propidium without invoking any conformational interaction between the peripheral and acylation sites.