Substrate Binding to the Peripheral Site of Acetylcholinesterase Initiates Enzymatic Catalysis. Substrate Inhibition Arises as a Secondary Effect

Abstract

Two sites of ligand interaction in acetylcholinesterase (AChE) were first demonstrated in ligand binding studies and later confirmed by crystallography, site-specific mutagenesis, and molecular modeling: an acylation site at the base of the active site gorge and a peripheral site at its mouth. We recently introduced a steric blockade model which demonstrated how small peripheral site ligands such as propidium may inhibit substrate hydrolysis [Szegletes, T., Mallender, W. D., and Rosenberry, T. L. (1998) Biochemistry 37, 4206-4216]. In this model, the only effect of a bound peripheral site ligand is to decrease the association and dissociation rate constants for an acylation site ligand without altering the equilibrium constant for ligand binding to the acylation site. Here, we first provide evidence that not only rate constants for substrates but also dissociation rate constants for their hydrolysis products are decreased by bound peripheral site ligand. Previous reaction schemes for substrate hydrolysis by AChE were extended to include product dissociation steps, and acetylthiocholine hydrolysis rates in the presence of propidium under nonequilibrium conditions were simulated with assigned rate constants in the program SCoP. We next showed that cationic substrates such as acetylthiocholine and 7-acetoxy-N-methylquinolinium (M7A) bind to the peripheral site as well as to the acylation site. The neurotoxin fasciculin was used to report specifically on interactions at the peripheral site. Analysis of inhibition of fasciculin association rates by these substrates revealed KS values of about 1 mM for the peripheral site binding of acetylthiocholine and 0.2 mM for the binding of M7A. The AChE reaction scheme was further extended to include substrate binding to the peripheral site as the initial step in the catalytic pathway. Simulations of the steric blockade model with this scheme were in reasonable agreement with observed substrate inhibition for acetylthiocholine and M7A and with mutual competitive inhibition in mixtures of acetylthiocholine and M7A. Substrate inhibition was explained by blockade of product dissociation when substrate is bound to the peripheral site. However, our analyses indicate that the primary physiologic role of the AChE peripheral site is to accelerate the hydrolysis of acetylcholine at low substrate concentrations.

Crystal Structure of Human Butyrylcholinesterase and of Its Complexes with Substrate and Products

“…Unexpectedly, the crystal structure of the Torpedo californica enzyme (TcAChE) showed that the active site catalytic Ser-His-Glu triad is found at the bottom of a 20-Å deep gorge lined mostly with aromatic residues (5). The structure also revealed the nature and the location of the previously described peripheral and "anionic" sites; the former, located at the outer rim of the gorge, has been postulated to be the initial substrate binding site (6). The binding of ligand to this site has been proposed to slow down the traffic of substrate and product at the acylation site (6,7).…”

Crystal Structure of a Lipoxygenase in Complex with Substrate

“…Substrate inhibition has been studied in other enzymes (20,(35)(36)(37), and its analysis can be complex (32,38,39). We observed that the R182A data are best explained by cooperative substrate inhibition (Equation 1).…”

Inhibitors Tethered Near the Acetylcholinesterase Active Site Serve as Molecular Rulers of the Peripheral and Acylation Sites

“…Assay solutions included 0.33 mM 5,5Ј-dithiobis(2-nitrobenzoic acid), and hydrolysis was monitored by formation of the thiolate dianion 3-carboxy-4-nitrothiophenol at 412 nm (⌬⑀ 412 nm ϭ 14.15 mM Ϫ1 cm Ϫ1 (29)). Hydrolysis rates v were measured at various substrate concentrations [S] in 20 mM sodium phosphate, 0.02% Triton X-100 (pH 7.0) at 25°C, and constant ionic strength was maintained with 0 -60 mM NaCl (14). …”

“…The peripheral site (P-site) consists of a binding pocket near the surface of the enzyme at the mouth of the gorge and specifically binds certain ligands like the neurotoxin fasciculin (8 -11) and the fluorescent probes propidium (12) and thioflavin T (13). We have recently shown that catalysis is accelerated because cationic substrates transiently bind to the P-site en route to the A-site (14,15). However, ligand binding to the P-site also can inhibit AChE through a process we have called steric blockade (15,16).…”