Catalytic itinerary in 1,3-1,4-beta-glucanase unraveled by QM/MM metadynamics. Charge is not yet fully developed at the oxocarbenium ion-like transition state

Xevi Biarnés, Albert Ardèvol, Javier Iglesias-Fernández, Antoni Planas, Carme Rovira

Research output: Indexed journal article Articlepeer-review

79 Citations (Scopus)

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 1,4B/1S3 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 (1,4B/1S 3 -4E/4H3 - 4C 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.

Original languageEnglish
Pages (from-to)20301-20309
Number of pages9
JournalJournal of the American Chemical Society
Volume133
Issue number50
DOIs
Publication statusPublished - 21 Dec 2011

Keywords

  • Free-energy landscape
  • Molecular-dynamics
  • Substrate distortion
  • Force-field
  • Mechanism
  • Glycosidase
  • Hydrolysis
  • Specificity
  • Insight
  • Initio

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