Organophosphorylation of Acetylcholinesterase in the Presence of Peripheral Site Ligands

Abstract

Structural analysis of acetylcholinesterase (AChE) has revealed two sites of ligand interaction in the active site gorge: an acylation site at the base of the gorge and a peripheral site at its mouth. A goal of our studies is to understand how ligand binding to the peripheral site alters the reactivity of substrates and organophosphates at the acylation site. Kinetic . Moreover, the binding of propidium resulted in a clear increase in k OP for EMPC, suggesting that molecular or electronic strain caused by the proximity of propidium to EMPC in the ternary complex may promote phosphorylation. In contrast, the binding of the polypeptide neurotoxin fasciculin to the peripheral site of AChE dramatically decreased phosphorylation rate constants. Values of k OP /K OP were decreased by factors of 10 3 to 10 5 , and k OP was decreased by factors of 300 -4,000. Such pronounced inhibition suggested a conformational change in the acylation site induced by fasciculin binding. As a note of caution to other investigators, measurements of phosphorylation of the fasciculin-AChE complex by AChE inactivation gave misleading rate constants because a small fraction of the AChE was resistant to inhibition by fasciculin. Acetylcholinesterase (AChE)1 terminates neurotransmission by catalyzing hydrolysis of the neurotransmitter acetylcholine at rates near that of a diffusion-controlled process (1). The x-ray crystal structure of AChE reveals that despite the impressive turnover rate of the enzyme, substrate molecules must penetrate 20 Å into a deep active site gorge to be hydrolyzed (2-4). This gorge contains two sites of ligand interaction: a peripheral site at the surface of the enzyme and an acylation site at the base of the gorge where the substrate acyl group is first transferred to residue Ser 200 (Torpedo californica AChE sequence numbering) and then hydrolyzed. In the acylation site, a catalytic triad consisting of residues Ser 200 , His 440 , and Glu 327 promotes the acyl transfers, and Trp 84 binds the acetylcholine trimethylammonium group, positioning the substrate for hydrolysis. Certain ligands can bind selectively to either the acylation site or the peripheral site, and ternary complexes can be formed in which ligands are bound to both sites simultaneously (5, 6). Ligands specific for the peripheral site include the small aromatic compound propidium and the snake venom neurotoxin fasciculin, both of which are potent inhibitors of the hydrolysis of the chromogenic acetylcholine analog, acetylthiocholine.The AChE peripheral site is an attractive target for the design of new classes of therapeutic agents, so it is important to understand how ligand binding to the peripheral site affects substrate hydrolysis. We recently provided evidence for a steric blockade model which proposes that small peripheral site ligands like propidium inhibit substrate hydrolysis by decreasing the association and dissociation rate constants for an acylation site ligand without significantly altering their ratio, the ligand equilibrium constant (7...

Thioflavin T Is a Fluorescent Probe of the Acetylcholinesterase Peripheral Site That Reveals Conformational Interactions between the Peripheral and Acylation Sites

“…However, 5-to 10-fold decreases in organophosphate affinity for propidium-bound AChE were observed with both a cationic and a neutral organophosphate, indicating an additional type of interaction (6,13). Molecular modeling calculations indicate that an organophosphate with a large leaving group would overlap with propidium if both ligands were placed in their expected sites in free AChE (13), and we have attributed these decreases in affinity to adjustments that accommodate both ligands in the ternary complex (6,13). Modeling calculations indicate that such steric overlap is not a factor in ternary complexes involving propidium-AChE and either huperzine A or TMTFA; yet ligand affinities in the ternary complexes also are 5-to 7-fold lower than in the corresponding binary complexes (17).…”

“…Steric blockade in our terminology is strictly a kinetic effect, and it can have no influence on substrate affinity or hydrolysis by slowly reacting substrates like organophosphates that equilibrate with AChE before acylation. However, 5-to 10-fold decreases in organophosphate affinity for propidium-bound AChE were observed with both a cationic and a neutral organophosphate, indicating an additional type of interaction (6,13). Molecular modeling calculations indicate that an organophosphate with a large leaving group would overlap with propidium if both ligands were placed in their expected sites in free AChE (13), and we have attributed these decreases in affinity to adjustments that accommodate both ligands in the ternary complex (6,13).…”

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

“…Recombinant human AChE was expressed as a secreted, disulfide-linked dimer in Drosophila S2 cells and purified as outlined previously (11). Thioflavin T and carbachol (carbamoylcholine chloride) were from Sigma.…”

Inhibitors of Different Structure Induce Distinguishing Conformations in the Omega Loop, Cys69–Cys96, of Mouse Acetylcholinesterase

“…Influence of Fasciculin Capping on the Spectrum of Phosphorylated and Carbamoylated AChEs-Because fasciculin is known to interact at the rim of the active center gorge of AChE (33)(34)(35) and can cap the gorge with a conjugated ligand at the base of the gorge (26,36), we examined the acrylodan spectra of the conjugated enzymes after fasciculin addition. Here again, the most informative position to analyze is 81.…”