Catalytic Itinerary in 1,3-1,4-尾-Glucanase Unraveled by QM/MM Metadynamics. Charge Is Not Yet Fully Developed at the Oxocarbenium Ion-like Transition State

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• 作者：Xevi Biarn茅s ; Albert Ard猫vol ; Javier Iglesias-Fern谩ndez ; Antoni Planas ; Carme Rovira
• 刊名：Journal of the American Chemical Society
• 出版年：2011
• 出版时间：December 21, 2011
• 年：2011
• 卷：133
• 期：50
• 页码：20301-20309
• 全文大小：1160K
• 年卷期：v.133,no.50(December 21, 2011)
• ISSN：1520-5126

文摘

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鈥揺nzyme 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-尾-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 鈭? 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/¹S₃ 鈥?sup>4E/⁴H₃ 鈥?⁴C₁) 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.