Acetylthiocholine binds to asp74 at the peripheral site of human acetylcholinesterase as the first step in the catalytic pathway

Studies of ligand binding to acetylcholinesterase (AChE) have demonstrated two sites of interaction. An acyl-enzyme intermediate is formed at the acyl ation 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 specif ic 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 cl arify the role of the peripheral site in the catalytic pathway for AChE, we showed that ligands which bind specifically to the peripheral site can slo w the rates at which other ligands enter and exit the acylation site, a fea ture we called steric blockade [Szegletes, T., Mallender, W. D., and Rosenb erry, T. L. (1998) Biochemistry 37, 4206-4216]. We also demonstrated that c ationic substrates can form a low-affinity complex at the peripheral site t hat accelerates catalytic hydrolysis at low substrate concentrations but re sults in substrate inhibition at high concentrations because of steric bloc kade of product release [Szegletes, T., Mallender, W. D., Thomas, P. J., an d Rosenberry, T. L. (1999) Biochemistry 38, 122-133]. In this report, we de monstrate that a key residue in the human AChE peripheral site with which t he substrate acetylthiocholine interacts is D74. We extend our kinetic mode l to evaluate the substrate affinity for the peripheral site, indicated by the equilibrium dissociation constant Ks, from the dependence of the substr ate hydrolysis rate on substrate concentration. For human AChE, a Ks of 1.9 +/- 0.7 mM obtained by fitting this substrate inhibition curve agreed with a Ks of 1.3 +/- 1.0 mM measured directly from acetylthiocholine inhibition of the binding of the neurotoxin fasciculin to the peripheral site. For To rpedo AChE, a Ks of 0.5 +/- 0.2 mM obtained from substrate inhibition agree d with a Ks of 0.4 +/- 0.2 mM measured with fasciculin. Introduction of the D72G mutation (corresponding to D74G in human AChE) increased the Ks to 4- 10 mM in the Torpedo enzyme and to about 33 mM in the human enzyme. While t he turnover number k(cat) was unchanged in the human D74G mutant, the rough ly 20-fold decrease in acetylthiocholine affinity for the peripheral site i n D74G resulted in a corresponding decrease in k(cat)/K-app, the second-ord er hydrolysis rate constant, in the mutant. In addition, we show that D74 i s important in conveying to the acylation site an inhibitory conformational effect induced by the binding of fasciculin to the peripheral site. This i nhibitory effect, measured by the relative decrease in the first-order phos phorylation rate constant k(OP) for the neutral organophosphate 7- [(methyl ethoxyphosphonyl)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.