title: Hydrolysis of low concentrations of the acetylthiocholine analogs acetyl(homo)thiocholine and acetyl(nor)thiocholine by acetylcholinesterase may be limited by selective gating at the enzyme peripheral site.
abstract: Hydrolysis of acetylcholine by acetylcholinesterase (AChE) is extremely rapid, with a second-order hydrolysis rate constant k(E) (often denoted k(cat)/K(M)) that approaches 10(8) M(-1) s(-1). AChE contains a deep active site gorge with two sites of ligand binding, an acylation site (or A-site) containing the catalytic triad at the base of the gorge and a peripheral site (or P-site) near the gorge entrance. The P-site is known to contribute to catalytic efficiency with acetylthiocholine (AcSCh) by transiently trapping the substrate in a low affinity complex on its way to the A-site, where a short-lived acyl enzyme intermediate is produced. Here we ask whether the P-site does more than simply trap the substrate but in fact selectively gates entry to the A-site to provide specificity for AcSCh (and acetylcholine) relative to the close structural analogs acetyl(homo)thiocholine (Ac-hSCh, which adds one additional methylene group to thiocholine) and acetyl(nor)thiocholine (Ac-nSCh, which deletes one methylene group from thiocholine). We synthesized Ac-hSCh and Ac-nSCh and overcame technical difficulties associated with instability of the northiocholine hydrolysis product. We then compared the catalytic parameters of these substrates with AChE to those of AcSCh. Values of k(E) for Ac-hSCh and Ac-nSCh were about 2% of that for AcSCh. The k(E) for AcSCh is close to the theoretical diffusion-controlled limit for the substrate association rate constant, but kE values for Ac-hSCh or Ac-nSCh are too low to be limited by diffusion control. However, analyses of kinetic solvent isotope effects and inhibition patterns for P-site inhibitors indicate that these two analogs also do not equilibrate with the A-site prior to the initial acylation step of catalysis. We propose that kE for these substrates is partially rate-limited by a gating step that involves the movement of bound substrate from the P-site to the A-site.
authors.affiliation: Mayo Clinic College of Medicine, Department of Neuroscience, Jacksonville, FL 32224, United States.
authors.forename: Veena
authors.initials: V
authors.lastname: Beri
authors.forename: Jeffrey T
authors.initials: JT
authors.lastname: Auletta
authors.forename: Ghulam M
authors.initials: GM
authors.lastname: Maharvi
authors.forename: Juanita F
authors.initials: JF
authors.lastname: Wood
authors.forename: Abdul H
authors.initials: AH
authors.lastname: Fauq
authors.forename: Terrone L
authors.initials: TL
authors.lastname: Rosenberry
mesh_terms: D000110:Acetylcholinesterase / Q000737:chemistry / Q000378:metabolism*; D000122:Acetylthiocholine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000215:Acylation; D020134:Catalytic Domain; D002800:Cholinesterase Inhibitors / Q000378:metabolism / Q000494:pharmacology; D058851:GPI-Linked Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D002800:Cholinesterase Inhibitors; D058851:GPI-Linked Proteins; D011994:Recombinant Proteins; D000122:Acetylthiocholine; C548244:ACHE protein, human; D000110:Acetylcholinesterase