The Conformational Free-Energy Landscape of β-D-Mannopyranose: Evidence for a ~1S_5 → B_(2,5) →~oS_2 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 ~1S_5 conformation, and recent experiments have proposed an unusual substrate conformational pathway (~1S_5 - B_(2,5) → °S_2) 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 ~1S_5 is among the most stable conformers and simultaneously is the most preactivated conformation in terms of elongation/shortening of the C1 -01/ C1-O5 bonds, C1-O1 orientation, and charge development at the anomeric carbon. Analysis of the computed FES gives support to the proposed ~1S_5→B_(2.5) →°S_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.