Unmasking tandem site interaction in human acetylcholinesterase. Substrate activation with a cationic acetanilide substrate.
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.
Johnson
Joseph L
JL
Department of Pharmacology, Mayo Foundation for Medical Education and Research, Mayo Clinic Jacksonville, Jacksonville, Florida 32224, USA.
Cusack
Bernadette
B
Davies
Matthew P
MP
Fauq
Abdul
A
Rosenberry
Terrone L
TL
D000083:Acetanilides / Q000378:metabolism*; D000098:Acetophenones / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D000110:Acetylcholinesterase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000122:Acetylthiocholine / Q000378:metabolism*; D000215:Acylation; D052160:Benzothiazoles; D001665:Binding Sites; D002384:Catalysis; D002800:Cholinesterase Inhibitors / Q000378:metabolism; D004347:Drug Interactions; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D008024:Ligands; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D011419:Propidium / Q000737:chemistry; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity / Q000187:drug effects; D013844:Thiazoles / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology
D000083:Acetanilides; D000098:Acetophenones; D052160:Benzothiazoles; D002800:Cholinesterase Inhibitors; D005456:Fluorescent Dyes; D008024:Ligands; D011994:Recombinant Proteins; D013844:Thiazoles; C111602:m-(N,N,N-trimethylammonio)trifluoroacetophenone; C009462:thioflavin T; D011419:Propidium; D000122:Acetylthiocholine; D000110:Acetylcholinesterase