The X-ray structure of a transition state analog complex reveals the molecular origins of the catalytic power and substrate specificity of acetylcholinesterase

The structure of a complex of Torpedo calfornica acetylcholinesterase with the transition state analog inhibitor m-(N,N,N-trimethylammonio)-2,2,2-trifluoroacetophenone has been solved by X-ray crystallographic methods to 2.8 Angstrom resolution. Since the inhibitor binds to the enzyme about 10(10)-fold more tightly than the substrate acetylcholine, this complex provides a visual accounting of the enzyme-ligand interactions that provide the molecular basis for the catalytic power of acetylcholinesterase. The enzyme owes about 8 kcal mol(-1) of the 18 kcal mol(-1) of free energy of stabilization of the acylation transition state to interactions of the quaternary ammonium moiety with three water molecules, with the carboxylate side chain of E199, and with the aromatic side chains of W84 and F330. The carbonyl carbon of the trifluoroketone function interacts covalently with S200 of the S200-H440-E327 catalytic triad. The operation of this triad as a general acid-base catalytic network probably provides 3-5 kcal mol(-1) of the free energy of stabilization of the transition state. The remaining 5-7 kcal mol(-1) of transition state stabilization probably arises from tripartite hydrogen bonding between the incipient oxyanion and the NH functions of G118, G119, and A201. The acetyl ester hydrolytic specificity of the enzyme is revealed by the interaction of the CF3 function of the transition state analog with a concave binding site comprised of the residues G119, W233, F288, F290, and F331. The highly geometrically convergent array of enzyme-ligand interactions visualized in the complex described herein envelopes the acylation transition state and sequesters it from solvent, this being consistent with the location of the active site at the bottom of a deep and narrow gorge.