A steric blockade model for inhibition of acetylcholinesterase by peripheral site ligands and substrate.
The active site gorge of acetylcholinesterase (AChE) contains two sites of ligand binding, an acylation site near the base of the gorge and a peripheral site at its mouth. We recently introduced a steric blockade model which demonstrated that small peripheral site ligands like propidium can inhibit substrate hydrolysis simply by decreasing the substrate association and dissociation rate constants without altering the equilibrium constant for substrate binding to the acylation site. We now employ our nonequilibrium kinetic analysis to extend this model to include blockade of the dissociation of substrate hydrolysis products by bound peripheral site ligand. We also report here that acetylthiocholine can bind to the AChE peripheral site with an equilibrium dissociation constant K(S) of about 1 mM. This value was determined from the effect of the acetylthiocholine concentration on the rate at which fasciculin associates with the peripheral site. When substrate binding to the peripheral site is incorporated into our steric blockade model, hydrolysis rates at low substrate concentration appear to be accelerated while substrate inhibition of hydrolysis occurs at high substrate concentration. The model predicts that hydrolysis rates for substrates which equilibrate with the acylation site prior to the acylation step should not be inhibited by bound peripheral site ligand. Organophosphates equilibrate with AChE prior to phosphorylating the active site serine residue, and as predicted propidium had little effect on the phosphorylation rate constants for the fluorogenic organophosphate ethylmethyl-phosphonylcoumarin (EMPC). The 2nd-order phosphorylation rate constant kOP/K(OP) was decreased 3-fold by a high concentration of propidium and the 1st-order rate constant kOP increased somewhat. In contrast to propidium, when the neurotoxin fasciculin bound to the AChE peripheral site both a steric blockade and a conformational change in the acylation site appeared to occur. With saturating fasciculin, kOP/K(OP) decreased by a factor of more than 750 and kOP decreased 300-fold. These data suggest that new peripheral site ligands may be designed to have selective effects on AChE phosphorylation.
Department of Pharmacology, Mayo Clinic Jacksonville, FL 32224, USA. rosenb@mayo.edu
T L
TL
Rosenberry
W D
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Mallender
P J
PJ
Thomas
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T
Szegletes
D000109:Acetylcholine / Q000378:metabolism; D000110:Acetylcholinesterase / Q000097:blood / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002800:Cholinesterase Inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D006868:Hydrolysis; D007202:Indicators and Reagents / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D008956:Models, Chemical; D010766:Phosphorylation; D011419:Propidium / Q000378:metabolism / Q000494:pharmacology; D011487:Protein Conformation; D013237:Stereoisomerism; D013379:Substrate Specificity
D002800:Cholinesterase Inhibitors; D007202:Indicators and Reagents; D008024:Ligands; D011419:Propidium; D000110:Acetylcholinesterase; D000109:Acetylcholine