Distinct roles of monosaccharides in the inhibition of the carbohydrate‐binding module and catalytic domain of Cel7A

Abstract The enzymatic breakdown of lignocellulosic biomass is a critical step toward the sustainable production of biochemicals. The cellobiohydrolase Cel7A plays a key role in this process by hydrolyzing cellulose. Both the primary reaction product (cellobiose) and secondary monosaccharides (glucose, mannose, and xylose) inhibit Cel7A's activity by interacting with its carbohydrate‐binding module (CBM) and catalytic domain (CD). Previous works have established that while glucose directly slows the enzyme's catalytic rate ( k cat ), mannose and xylose primarily reduce its association rate ( k on ) and overall processivity. To uncover the microscopic mechanisms behind these observations, we employed all‐atom molecular dynamics simulations combined with the Kirkwood–Buff solution theory. Our results reveal an unsuspected contrast in the solvation preferences of the two domains: while sugars are preferentially excluded from the CD, the CBM exhibits preferential sugar binding. We observed a high density of sugars along the CBM's planar binding face (residues Y5, Y31, and Y32), as well as near I11, S14, S21, V27, L28, and P30. Xylose, the most potent experimental inhibitor, displays the highest density near the CBM residues comprising the cellulose‐binding face, which could interfere with the enzyme's anchoring of cellulose. Furthermore, we observed sugar accumulation at the entrance of the CD tunnel and within the tunnel‐binding loops (B2, B3, and A3), providing a structural explanation for the obstruction of the enzyme's processive cycle. By reconciling broader solvation preferences with highly specific site interactions, our findings offer a picture of how glucose and hemicellulose‐derived sugars inhibit Cel7A.