Abstract
The structure and dynamics of the enzyme-substrate complex of Bacillus 1,3-1,4-beta-glucanase, one of the most active glycoside hydrolases, is investigated by means of Car-Parrinello molecular dynamics simulations (CPMD) combined with force field molecular dynamics (QM/MM CPMD). It is found that the substrate sugar ring located at the -1 subsite adopts a distorted 1S3 skew-boat conformation upon binding to the enzyme. With respect to the undistorted 4C1 chair conformation, the 1S3 skew-boat conformation is characterized by: (a) an increase of charge at the anomeric carbon (C1), (b) an increase of the distance between C1 and the leaving group, and (c) a decrease of the intraring O5-C1 distance. Therefore, our results clearly show that the distorted conformation resembles both structurally and electronically the transition state of the reaction in which the substrate acquires oxocarbenium ion character, and the glycosidic bond is partially broken. Together with analysis of the substrate conformational dynamics, it is concluded that the main determinants of substrate distortion have a structural origin. To fit into the binding pocket, it is necessary that the aglycon leaving group is oriented toward the beta region, and the skew-boat conformation naturally fulfills this premise. Only when the aglycon is removed from the calculation the substrate recovers the all-chair conformation, in agreement with the recent determination of the enzyme product structure. The QM/MM protocol developed here is able to predict the conformational distortion of substrate binding in glycoside hydrolases because it accounts for polarization and charge reorganization at the -1 sugar ring. It thus provides a powerful tool to model E.S complexes for which experimental information is not yet available.
Highlights
The structure and dynamics of the enzyme-substrate complex of Bacillus 1,3–1,4--glucanase, one of the most active glycoside hydrolases, is investigated by means of Car-Parrinello molecular dynamics simulations (CPMD) combined with force field molecular dynamics (QM/MM CPMD)
A current issue in the understanding of Glycoside hydrolases (GHs) mechanisms is the conformational itinerary that the substrate follows during the reaction [5, 6], in which substrate distortion is induced upon binding to the enzyme to reach a transition state with sp2 geometry at the anomeric carbon
The substrate conformation in the Michaelis complex of GHs influences the conformational itinerary of the substrate during the catalytic reaction [5, 6]; it is a topic of ongoing interest in glycobiology
Summary
The structure and dynamics of the enzyme-substrate complex of Bacillus 1,3–1,4--glucanase, one of the most active glycoside hydrolases, is investigated by means of Car-Parrinello molecular dynamics simulations (CPMD) combined with force field molecular dynamics (QM/MM CPMD). The E1⁄7S Complex of 1,3–1,4--Glucanase tides by NMR techniques [26] This kind of saccharide ring distortion has favorable mechanistic consequences in glycoside hydrolysis. The 1S3 distortion places the glycosidic oxygen near the acid/base residue It reduces the steric interaction between the hydrogen at the anomeric carbon and the nucleophile, and places the aglycon (i.e. the leaving group) in a pseudo-axial position that facilitates nucleophile attack on the anomeric carbon [27]. These distortions in the Michaelis complex are in the pathway to reach the transition state of the reaction. In most cases these distortions are encountered in inhibitor-enzyme complexes (i.e. modified forms of the substrate) or in complexes with inactive enzyme mutants, which raises the question whether the inhibitor or the mutant could affect the substrate conformation [28]
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