|Title||The Mechanism of Cellulose Hydrolysis by a Two-Step, Retaining Cellobiohydrolase Elucidated by Structural and Transition Path Sampling Studies|
|Publication Type||Journal Article|
|Year of Publication||2014|
|Authors||Knott, BC, Momeni, MHaddad, Crowley, MF, Mackenzie, LF, Goetz, AW, Sandgren, M, Withers, SG, Stahlberg, J, Beckham, GT|
|Journal||JOURNAL OF THE AMERICAN CHEMICAL SOCIETY|
|Date Published||JAN 8|
Glycoside hydrolases (GHs) cleave glycosidic linkages in carbohydrates, typically via inverting or retaining mechanisms, the latter of which proceeds via a two-step mechanism that includes formation of a glycosyl-enzyme intermediate. We present two new structures of the catalytic domain of Hypocrea jecorina GH Family 7 cellobiohydrolase Cel7A, namely a Michaelis complex with a full cellononaose ligand and a glycosyl-enzyme intermediate, that reveal details of the `static' reaction coordinate. We also employ transition path sampling to determine the `dynamic' reaction coordinate for the catalytic cycle. The glycosylation reaction coordinate contains components of forming and breaking bonds and a conformational change in the nucleophile. Deglycosylation proceeds via a product-assisted mechanism wherein the glycosylation product, cellobiose, positions a water molecule for nucleophilic attack on the anomeric carbon of the glycosyl-enzyme intermediate. In concert with previous structures, the present results reveal the complete hydrolytic reaction coordinate for this naturally and industrially important enzyme family.