Catalytic reaction mechanism of acetylcholinesterase determined by Born-Oppenheimer ab initio QM/MM molecular dynamics simulations

Abstract

Acetylcholinesterase (AChE) is a remarkably efficient serine hydrolase responsible for the termination of impulse signaling at cholinergic synapses. By employing Born-Oppenheimer molecular dynamics simulations with a B3LYP/6-31G(d) QM/MM potential and the umbrella sampling method, we have characterized its complete catalytic reaction mechanism for hydrolyzing neurotransmitter acetylcholine (ACh) and determined its multistep free-energy reaction profiles for the first time. In both acylation and deacylation reaction stages, the first step involves the nucleophilic attack on the carbonyl carbon, with the triad His447 serving as the general base, and leads to a tetrahedral covalent intermediate stabilized by the oxyanion hole. From the intermediate to the product, the orientation of the His447 ring needs to be adjusted very slightly, and then, the proton transfers from His447 to the product, and the break of the scissile bond happens spontaneously. For the three-pronged oxyanion hole, it only makes two hydrogen bonds with the carbonyl oxygen at either the initial reactant or the final product state, but the third hydrogen bond is formed and stable at all transition and intermediate states during the catalytic process. At the intermediate state of the acylation reaction, a short and low-barrier hydrogen bond (LBHB) is found to be formed between two catalytic triad residues His447 and Glu334, and the spontaneous proton transfer between two residues has been observed. However, it is only about 1-2 kcal/mol stronger than the normal hydrogen bond. In comparison with previous theoretical investigations of the AChE catalytic mechanism, our current study clearly demonstrates the power and advantages of employing Born-Oppenheimer ab initio QM/MM MD simulations in characterizing enzyme reaction mechanisms.

Figure 1

Illustration of the AChE-ACh complex. (a) Cartoon representation of the enzyme and stick representation of the active site, (b) stick representation of important residues.

Figure 2

Reaction mechanism of ACh hydrolysis catalyzed by AChE.

Figure 3

Free energy profile for (a) the acylation reaction stage, (b) the deacylation reaction stage determined by B3LYP(6-31G*) QM/MM molecular dynamics simulations and umbrella sampling.

Figure 4

Illustration of the structures for the characterized acylation reaction stage. ES, enzyme-substrate complex; TS1, the first transition state; TI1, tetrahedral intermediate; TS1′, the second transition state in acylation; EA1, acylated enzyme. Two representative configurations of TI1 are shown.

Figure 5

Calculated distances R_N-H (the distance between HD1 and ND1 of His447) and R_O-H (distance between HD1 and carboxyl oxygen OE1 of Glu334) in QM/MM-MD simulation at the intermediate TI1.

Figure 6

Illustration of the structures for the characterized deacylation reaction stage. EA2, acylated enzyme in deacylaton; TS2, first transition state; TI2, tetrahedral intermediate; TS2′, the second transition state; EP, enzyme-product complex.

Figure 7

(a) Free energy profile for the proton transfer between Glu334 and His447 at the tetrahedral intermediate state (TI1) of the acylation stage. The reaction coordinate is R_ND1-HD1 – R_OE1-HD1. From left to right, the proton transfer from His447 to the carboxyl oxygen of Glu334. (b) Free energy profile for the elongation of H-bond formed between Glu334 and His447 at ES state and TI1 state. Reaction coordinate is the distance between the carboxyl oxygen atom OE1 of Glu334 and the nitrogen atom ND1 of His447.