The Mechanism of Cellulose Hydrolysis by a Two-Step, Retaining Cellobiohydrolase Elucidated by Structural and Transition Path Sampling Studies

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.