The reaction mechanism of acetylcholine hydrolysis by acetylcholinesterase, including both acylation and deacylation stages from the enzyme-substrate (ES) to the enzyme-product (EP) molecular complexes, is examined by using an ab initio type quantum mechanical - molecular mechanical (QM/MM) approach. The density functional theory PBE0/aug-6-31+G* method for a fairly large quantum part trapped inside the native protein environment, and the AMBER force field parameters in the molecular mechanical part are employed in computations. All reaction steps, including the formation of the first tetrahedral intermediate (TI1), the acylenzyme (EA) complex, the second tetrahedral intermediate (TI2), and the EP complex, are modeled at the same theoretical level. In agreement with the experimental rate constants, the estimated activation energy barrier of the deacylation stage is slightly higher than that for the acylation phase. The critical role of the non-triad Glu202 amino acid residue in orienting lytic water molecule and in stabilizing the second tetrahedral intermediate at the deacylation stage of the enzymatic process is demonstrated.

Comparison and multiple alignments of amino acid sequences of a representative number of related enzymes demonstrate the existence of certain positions of amino acid residues which are permanently reproducible in all members of the whole family. The use of the bioinformatic approach revealed conservative residues in each of the related enzymes and ranked amino acid conservatism for the overall enzymatic catalysis. Glycine and aspartic acid residues were shown to be the most essential for structure and catalytic activity of enzymes. Amino acid residues forming catalytic subsite of the active site of enzymes are always highly conservative. Analysis revealed that aspartic acid carboxyl group is the most frequently employed nucleophilic (in deprotonated form) and electrophilic (in protonated form) agent involved in activation of molecules by the mechanism of general base and acidic catalyses in the catalytic sites of enzymes. Glycine is a unique amino acid possessing the highest possibilities for rotation along C-C and C-N bonds of the polypeptide chain. The conservative fixation of the glycine residue in polypeptide chains of related enzymes provides a possibility for directed assembly of amino acid residues into the catalytic subsite structure. It is possible that the conservative glycines provide known conformational mobility of the protein and the active site. Methods of molecular modeling were used for analysis of structural substitutions of conservative and non-conservative glycines and their effects on geometry of catalytic site of typical hydrolases. The substitution of glycine(s) for alanine significantly altered the catalytic site structures.

The complete catalytic cycle of aspartoacylase (ASPA), a zinc-dependent enzyme responsible for cleavage of N-acetyl-l-aspartate, is characterized by the methods of molecular modeling. The reaction energy profile connecting the enzyme-substrate (ES) and the enzyme-product (EP) complexes is constructed by the quantum mechanics/molecular mechanics (QM/MM) method assisted by the molecular dynamics (MD) simulations with the QM/MM potentials. Starting from the crystal structure of ASPA complexed with the intermediate analogue, the minimum-energy geometry configurations and the corresponding transition states are located. The stages of substrate binding to the enzyme active site and release of the products are modeled by MD calculations with the replica-exchange umbrella sampling technique. It is shown that the first reaction steps, nucleophilic attack of a zinc-bound nucleophilic water molecule at the carbonyl carbon and the amide bond cleavage, are consistent with the glutamate-assisted mechanism hypothesized for the zinc-dependent hydrolases. The stages of formation of the products, acetate and l-aspartate, and regeneration of the enzyme are characterized for the first time. The constructed free energy diagram from the reactants to the products suggests that the enzyme regeneration, but not the nucleophilic attack of the catalytic water molecule, corresponds to the rate-determining stage of the full catalytic cycle of ASPA.