title: Catalytic itinerary in 1,3-1,4-β-glucanase unraveled by QM/MM metadynamics. Charge is not yet fully developed at the oxocarbenium ion-like transition state.
abstract: Retaining glycoside hydrolases (GHs), key enzymes in the metabolism of polysaccharides and glycoconjugates and common biocatalysts used in chemoenzymatic oligosaccharide synthesis, operate via a double-displacement mechanism with the formation of a glycosyl-enzyme intermediate. However, the degree of oxocarbenium ion character of the reaction transition state and the precise conformational itinerary of the substrate during the reaction, pivotal in the design of efficient inhibitors, remain elusive for many GHs. By means of QM/MM metadynamics, we unravel the catalytic itinerary of 1,3-1,4-β-glucanase, one of the most active GHs, belonging to family 16. We show that, in the Michaelis complex, the enzyme environment restricts the conformational motion of the substrate to stabilize a (1,4)B/(1)S(3) conformation of the saccharide ring at the -1 subsite, confirming that this distortion preactivates the substrate for catalysis. The metadynamics simulation of the enzymatic reaction captures the complete conformational itinerary of the substrate during the glycosylation reaction ((1,4)B/(1)S(3) -(4)E/(4)H(3) - (4)C(1)) and shows that the transition state is not the point of maximum charge development at the anomeric carbon. The overall catalytic mechanism is of dissociative type, and proton transfer to the glycosidic oxygen is a late event, clarifying previous kinetic studies of this enzyme.
authors: Computer Simulation and Modeling Laboratory, Parc Científic de Barcelona, Baldiri Reixac 10-12, 08028 Barcelona, Spain.
authors: Xevi
authors: X
authors: Biarnés
authors: Albert
authors: A
authors: Ardèvol
authors: Javier
authors: J
authors: Iglesias-Fernández
authors: Antoni
authors: A
authors: Planas
authors: Carme
authors: C
authors: Rovira
mesh_terms: D002384:Catalysis; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation; D011789:Quantum Theory; D013379:Substrate Specificity
chemical_list: D006026:Glycoside Hydrolases