Unmasking Tandem Site Interaction in Human Acetylcholinesterase. Substrate Activation with a Cationic Acetanilide Substrate

Abstract

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 catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. A conformational interaction between the A- and P-sites has recently been found to modulate ligand affinities. We now demonstrate that this interaction is of functional importance by showing that the acetylation rate constant of a substrate bound to the A-site is increased by a factor a when a second molecule of substrate binds to the P-site. This demonstration became feasible through the introduction of a new acetanilide substrate analogue of acetylcholine, 3-(acetamido)-N,N,N-trimethylanilinium (ATMA), for which a = 4. This substrate has a low acetylation rate constant and equilibrates with the catalytic site, allowing a tractable algebraic solution to the rate equation for substrate hydrolysis. ATMA affinities for the A- and P-sites deduced from the kinetic analysis were confirmed by fluorescence titration with thioflavin T as a reporter ligand. Values of a >1 give rise to a hydrolysis profile called substrate activation, and the AChE site-specific mutant W86F, and to a lesser extent wild-type human AChE itself, showed substrate activation with acetylthiocholine as the substrate. Substrate activation was incorporated into a previous catalytic scheme for AChE in which a bound P-site ligand can also block product dissociation from the A-site, and two additional features of the AChE catalytic pathway were revealed. First, the ability of a bound P-site ligand to increase the substrate acetylation rate constant varied with the structure of the ligand: thioflavin T accelerated ATMA acetylation by a factor a(2) of 1.3, while propidium failed to accelerate. Second, catalytic rate constants in the initial intermediate formed during acylation (EAP, where EA is the acyl enzyme and P is the alcohol leaving group cleaved from the ester substrate) may be constrained such that the leaving group P must dissociate before hydrolytic deacylation can occur.

The biological significance of substrate inhibition: A mechanism with diverse functions

“…Salpeter and coworkers [26,27] and Rosenberry and coworkers [28,29] have carried out detailed studies of the kinetics of ACH release, binding to post-synaptic receptors, and degradation by ACHe.…”

Monitoring the reaction of carbachol with acetylcholinesterase by thioflavin T fluorescence and acetylthiocholine hydrolysis

“…1 to obtain values of the overall rate constants k 12 and k 21 (14). However, slightly better fits were obtained when the rate equations corresponding to Scheme 1 were solved directly by the numerical integration program SCoP (Simulation Resources, Inc., Redlands, CA; version 3.52) that we have used previously (12,17). This program allows ligand concentrations to vary over the reaction time course, and it thereby permits analysis at low concentrations of S 2 to minimize the contributions of terms with S 2 to expressions for k 12 and k 21 .…”

“…However, slightly better fits were obtained when the rate equations corresponding to Scheme 1 were solved directly by the numerical integration program SCoP (Simulation Resources, Inc., Redlands, CA; version 3.52) that we have used previously (12,17). This program allows ligand concentrations to vary over the reaction time course, and it thereby permits analysis at low concentrations of S 2 to minimize the contributions of terms with S 2 to expressions for k 12 and k 21 . These expressions, which depend on derivations of L and N for a selected reaction scheme like Scheme 2 below (e.g., 14), provide much mechanistic insight but will not be considered further in this report.…”

“…In experiments with thioflavin T and AChE alone, the fluorescence (F) was recorded over 3 -4 min intervals in the Hi-Tech SFA 20 stopped flow apparatus described above for improved F stability. Average F values were corrected for inner filter effects (18) as described previously (12). Data were consistent with a single thioflavin T binding site on AChE and analyzed with eq.…”

Synthesis and in silico evaluation of 1N-methyl-1S-methyl-2-nitroethylene (NMSM) derivatives against Alzheimer disease: to understand their interacting mechanism with acetylcholinesterase

“…Besides the CT and CAS, the residues of peripheral anionic site (PAS; Tyr72, Asp74, Tyr124, Trp286, and Tyr341) also influence the function of AChE through their allosteric effects [16,20,21]. Residues of PAS trap the substrate molecules as they enter the gorge, and are involved in accelerating the transfer of the substrate acyl group to a serine hydroxyl group in CAS [17,18,[22][23][24][25]. In addition, PAS site is potentially responsible for colocalization of amyloid-β (Aβ) peptide in the brains of AD patients and has been shown to accelerate the assembly of Aβ to form amyloid fibrils [26][27][28].…”