Molecular basis of inhibition of substrate hydrolysis by a ligand bound to the peripheral site of acetylcholinesterase.

Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to the catalytic efficiency of substrate hydrolysis by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. Ligands that bind to the A-site invariably inhibit the hydrolysis of all AChE substrates, but ligands that bind to the P-site inhibit the hydrolysis of some substrates but not others. To clarify the basis of this difference, we focus here on second-order rate constants for substrate hydrolysis (k(E)), a parameter that reflects the binding of ligands only to the free form of the enzyme and not to enzyme-substrate intermediates. We first describe an inhibitor competition assay that distinguishes whether a ligand is inhibiting AChE by binding to the A-site or the P-site. We then show that the P-site-specific ligand thioflavin T inhibits the hydrolysis of the rapidly hydrolyzed substrate acetylthiocholine but fails to show any inhibition of the slowly hydrolyzed substrates ATMA (3-(acetamido)-N,N,N-trimethylanilinium) and carbachol. We derive an expression for k(E) that accounts for these observations by recognizing that the rate-limiting steps for these substrates differ. The rate-limiting step for the slow substrates is the general base-catalyzed acylation reaction k(2), a step that is unaffected by bound thioflavin T. In contrast, the rate-limiting step for acetylthiocholine is either substrate association or substrate migration to the A-site, and these steps are blocked by bound thioflavin T.