The Conformational Free-Energy Landscape of β-D-Mannopyranose: Evidence for a 1S5 → B2,5 → OS2 Catalytic Itinerary in β-Mannosidases

The mechanism of glycosidic bond cleavage by glycosidases involves substrate ring distortions in the Michaelis complex that favor catalysis. Retaining beta-mannosidases bind the substrate in a S-1(5) conformation, and recent experiments have proposed an unusual substrate conformational pathway (S-1(5) -> B-2,B-5 -> S-O(2)) for the hydrolysis reaction. By means of Car-Parrinello metadynamics simulations, we have obtained the conformational free-energy surface (FES) of a beta-D-mannopyranose molecule associated with the ideal Stoddart conformational diagram. We have found that S-1(5) is among the most stable conformers and simultaneously is the most preactivated conformation in terms of elongation/shortening of the C1-O1/C1-O5 bonds, C1-O1 orientation, and charge development at the anomeric carbon. Analysis of the computed FES gives support to the proposed S-1(5) -> B-2,B-5 -> S-O(2) catalytic itinerary, showing that the degree of preactivation of the substrate in glycoside hydrolases (GHs) is related to the properties of an isolated sugar ring. We introduce a simple preactivation index integrating several structural, electronic, and energetic properties that can be used to predict the conformation of the substrate in the Michaelis complex of any GH.