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

摘要

The mechanism of glycosidic bond cleavage by glycosidases involves substrate ring distortions in the Michaelis complex that favor catalysis. Retaining β-mannosidases bind the substrate in a 1S₅ conformation, and recent experiments have proposed an unusual substrate conformational pathway (1S₅ → B_2,5 → OS₂) for the hydrolysis reaction. By means of Car−Parrinello metadynamics simulations, we have obtained the conformational free-energy surface (FES) of a β-d-mannopyranose molecule associated with the ideal Stoddart conformational diagram. We have found that 1S₅ 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 1S₅ → B_2,5 → OS₂ 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.