Acetylthiocholine Binds to Asp74 at the Peripheral Site of Human Acetylcholinesterase as the First Step in the Catalytic Pathway

Abstract

Studies of ligand binding to acetylcholinesterase (AChE) have demonstrated two sites of interaction. An acyl-enzyme intermediate is formed at the acylation site, and catalytic activity can be inhibited by ligand binding to a peripheral site. The three-dimensional structures of AChE-ligand complexes reveal a narrow and deep active site gorge and indicate that ligands specific for the acylation site at the base of the gorge must first traverse the peripheral site near the gorge entrance. In recent studies attempting to clarify the role of the peripheral site in the catalytic pathway for AChE, we showed that ligands which bind specifically to the peripheral site can slow the rates at which other ligands enter and exit the acylation site, a feature we called steric blockade [Szegletes, T., Mallender, W. D., and Rosenberry, T. L. (1998) Biochemistry 37, 4206-4216]. We also demonstrated that cationic substrates can form a low-affinity complex at the peripheral site that accelerates catalytic hydrolysis at low substrate concentrations but results in substrate inhibition at high concentrations because of steric blockade of product release [Szegletes, T., Mallender, W. D., Thomas, P. J., and Rosenberry, T. L. (1999) Biochemistry 38, 122-133]. In this report, we demonstrate that a key residue in the human AChE peripheral site with which the substrate acetylthiocholine interacts is D74. We extend our kinetic model to evaluate the substrate affinity for the peripheral site, indicated by the equilibrium dissociation constant K(S), from the dependence of the substrate hydrolysis rate on substrate concentration. For human AChE, a K(S) of 1.9+/-0.7 mM obtained by fitting this substrate inhibition curve agreed with a K(S) of 1.3+/-1.0 mM measured directly from acetylthiocholine inhibition of the binding of the neurotoxin fasciculin to the peripheral site. For Torpedo AChE, a K(S) of 0.5+/- 0.2 mM obtained from substrate inhibition agreed with a K(S) of 0.4+/- 0.2 mM measured with fasciculin. Introduction of the D72G mutation (corresponding to D74G in human AChE) increased the K(S) to 4-10 mM in the Torpedo enzyme and to about 33 mM in the human enzyme. While the turnover number k(cat) was unchanged in the human D74G mutant, the roughly 20-fold decrease in acetylthiocholine affinity for the peripheral site in D74G resulted in a corresponding decrease in k(cat)/K(app), the second-order hydrolysis rate constant, in the mutant. In addition, we show that D74 is important in conveying to the acylation site an inhibitory conformational effect induced by the binding of fasciculin to the peripheral site. This inhibitory effect, measured by the relative decrease in the first-order phosphorylation rate constant k(OP) for the neutral organophosphate 7-[(methylethoxyphosphonyl)oxy]-4-methylcoumarin (EMPC) that resulted from fasciculin binding, decreased from 0.002 in wild-type human AChE to 0.24 in the D74G mutant.

Crystal structure of N,N′-dibenzyl-N,N′-dimethyl- N″-(2-fluorobenzoyl)phosphoric diamide, C23H25FN3O2P

“…Carbacylamidophosphatesare attractive to study owing to their extensive applications in biochemistry [1,2]. These compounds are also used aschelating reagents for various metalions [3][4][5].…”

Inhibitors Tethered Near the Acetylcholinesterase Active Site Serve as Molecular Rulers of the Peripheral and Acylation Sites

“…Materials-Recombinant human wild type and H287C mutant AChEs were expressed as secreted dimeric forms in Drosophila S2 cells in culture (15,19) and purified by two cycles of affinity chromatography on acridinium resin (20). The mutant AChE construction and expression methods were similar to that described previously (21).…”

“…[E] tot values for both wild type and unmodified H287C AChE were calculated by assuming 450 units/nmol (13,15), 3 and for radio-methylated AChE these [E] tot values were converted to dpm/nmol ratios that were also applied to the modified AChEs.…”

“…The peripheral site (P-site) consists of a binding pocket near the surface of the enzyme at the mouth of the gorge and specifically binds certain ligands like the neurotoxin fasciculin (8 -11) and the fluorescent probes propidium (12) and thioflavin T (13). We have recently shown that catalysis is accelerated because cationic substrates transiently bind to the P-site en route to the A-site (14,15). However, ligand binding to the P-site also can inhibit AChE through a process we have called steric blockade (15,16).…”

“…We have recently shown that catalysis is accelerated because cationic substrates transiently bind to the P-site en route to the A-site (14,15). However, ligand binding to the P-site also can inhibit AChE through a process we have called steric blockade (15,16). This process involves a decrease in the rate constants with which substrates and their hydrolysis products enter and exit the A-site.…”