An Epoxide Intermediate in Glycosidase Catalysis
AuthorSobala, LF; Speciale, G; Zhu, S; Raich, L; Sannikova, N; Thompson, AJ; Hakki, Z; Lu, D; Abadi, SSK; Lewis, AR; ...
Source TitleACS Central Science
PublisherAMER CHEMICAL SOC
University of Melbourne Author/sWilliams, Spencer
AffiliationSchool of Chemistry
Document TypeJournal Article
CitationsSobala, L. F., Speciale, G., Zhu, S., Raich, L., Sannikova, N., Thompson, A. J., Hakki, Z., Lu, D., Abadi, S. S. K., Lewis, A. R., Rojas-Cervellera, V., Bernardo-Seisdedos, G., Zhang, Y., Millet, O., Jimenez-Barbero, J., Bennet, A. J., Sollogoub, M., Rovira, C., Davies, G. J. & Williams, S. J. (2020). An Epoxide Intermediate in Glycosidase Catalysis. ACS CENTRAL SCIENCE, 6 (5), pp.760-770. https://doi.org/10.1021/acscentsci.0c00111.
Access StatusAccess this item via the Open Access location
Open Access URLPublished version
Retaining glycoside hydrolases cleave their substrates through stereochemical retention at the anomeric position. Typically, this involves two-step mechanisms using either an enzymatic nucleophile via a covalent glycosyl enzyme intermediate or neighboring-group participation by a substrate-borne 2-acetamido neighboring group via an oxazoline intermediate; no enzymatic mechanism with participation of the sugar 2-hydroxyl has been reported. Here, we detail structural, computational, and kinetic evidence for neighboring-group participation by a mannose 2-hydroxyl in glycoside hydrolase family 99 endo-α-1,2-mannanases. We present a series of crystallographic snapshots of key species along the reaction coordinate: a Michaelis complex with a tetrasaccharide substrate; complexes with intermediate mimics, a sugar-shaped cyclitol β-1,2-aziridine and β-1,2-epoxide; and a product complex. The 1,2-epoxide intermediate mimic displayed hydrolytic and transfer reactivity analogous to that expected for the 1,2-anhydro sugar intermediate supporting its catalytic equivalence. Quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar via a transition state in an unusual flattened, envelope (E 3) conformation. Kinetic isotope effects (k cat/K M) for anomeric-2H and anomeric-13C support an oxocarbenium ion-like transition state, and that for C2-18O (1.052 ± 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymatic mechanism.
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