Golgi α-mannosidase II (GMII), a member of glycoside hydrolase family 38, cleaves two mannosyl residues from GlcNAcMan₅GlcNAc₂ as part of the N-linked glycosylation pathway. To elucidate the molecular and electronic details of the reaction mechanism, in particular the conformation of the substrate at the transition state, we performed quantum mechanics/molecular mechanics metadynamics simulations of the glycosylation reaction catalyzed by GMII. The calculated free energy of activation for mannosyl glycosylation (23 kcal/mol) agrees very well with experiments, as does the conformation of the glycon mannosyl ring in the product of the glycosylation reaction (the covalent intermediate). In addition, we provide insight into the electronic aspects of the molecular mechanism that were not previously available. We show that the substrate adopts an ^OS₂/B_2,5 conformation in the GMII Michaelis complex and that the nucleophilic attack occurs before complete departure of the leaving group, consistent with a D_NA_N reaction mechanism. The transition state has a clear oxacarbenium ion (OCI) character, with the glycosylation reaction following an ^OS₂/B_2,5 → B_2,5 [TS] → ¹S₅ itinerary, agreeing with an earlier proposal based on comparing α- and β-mannanases. The simulations also demonstrate that an active-site Zn ion helps to lengthen the O2′−H_O2′ bond when the substrate acquires OCI character, relieving the electron deficiency of the OCI-like species. Our results can be used to explain the potency of recently formulated GMII anticancer inhibitors, and they are potentially relevant in deriving new inhibitors.

A detailed understanding of the catalytic strategy of cellulases is key to finding alternative ways to hydrolyze cellulose to mono-, di-, and oligosaccharides. Endoglucanases from glycoside hydrolase family 8 (GH8) catalyze the hydrolysis of β-1,4-glycosidic bonds in cellulose by an inverting mechanism believed to involve a oxacarbenium ion-like transition state (TS) with a boat-type conformation of the glucosyl unit in subsite −1. In this work, hydrolysis by Clostridium thermocellum endo-1,4-glucanase A was computationally simulated with quantum mechanics/molecular mechanics metadynamics based on density functional theory. Our calculations show that the glucosyl residue in subsite −1 in the Michaelis complex is in a distorted ²S_O/^2,5B ring conformation, agreeing well with its crystal structure. In addition, our simulations capture the cationic oxacarbenium ion-like character of the TS with a partially formed double bond between the ring oxygen and C5′ carbon atoms. They also provide previously unknown structural information of important states along the reaction pathway. The simulations clearly show for the first time in GH8 members that the TS features a boat-type conformation of the glucosyl unit in subsite −1. The overall catalytic mechanism follows a D_N*A_N-like mechanism and a β-²S_O → ^2,5B [TS] → α-⁵S₁ conformational itinerary along the reaction coordinate, consistent with the anti-periplanar lone pair hypothesis. Because of the structural similarities and sequence homology among all GH8 members, our results can be extended to all GH8 cellulases, xylanases, and other endoglucanases. In addition, we provide evidence supporting the role of Asp278 as the catalytic proton acceptor (general base) for GH8a subfamily members.