Use of Quantum Mechanics/Molecular Mechanics Metadynamics Computation to Determine Hydrolase Mechanisms

Determining the detailed mechanisms of enzyme-catalyzed reactions is a difficult task, and understanding the structure(s) of the one or more transition states is even more difficult. With the continuing rapid increase in computing power, we now have tools to attack this problem.

We have used quantum mechanics to model roughly 50 atoms covering much of the substrates and the side chains of the amino acid residues in the active sites of four hydro­lases – GH8 Clostridium thermocellum endo-1,4-glucanase A, GH38 Drosphilia melanogaster Golgi α-mannosidase II, GH43 Geobacillus stearothermophilus Xyn3 β-xylosidase, and TE8 human THEM2 acyl-CoA thioesterase. The rest of the four substrate and four enzyme molecules were modeled with molecular mechanics.

We used Car-Parrinello molecular dynamics and metadynamics to simulate the hydro­lytic reactions catalyzed by the four enzymes. The inverting endoglucanase and β-xylosid­ase first protonate the substrate with their nonreducing-side glucosyl or xylosyl residue distorted into a ^2,5B shape, followed by breakage of the β-glycosidic bond with nucleophilic attack of water on the C1’ atom. The limiting covalent-bond-forming reaction catalyzed by the retaining α-mannosidase features protonation of the glycosidic bond with attack of the enzyme nucleophilic carboxyl group on the C1’ atom before complete departure of the leaving group, with the nonreducing-side mannosyl residue in a B_2,5 shape. Finally, the HotDog-fold thioesterase uses a single-displacement acid/base-like mechanism to first protonate the sulfur atom by a serine residue activated by a histidine residue, with nucleo­philic attack by a water molecule activated by an aspartate residue on the carbonyl atom.