Role of Tryptophan 38 in Loading Substrate Chain into the Active-site Tunnel of Cellobiohydrolase I from Trichoderma reesei

Cellobiohydrolase I from Trichoderma reesei (TrCel7A) is one of the best-studied cellulases, exhibiting high activity towards crystalline cellulose. Tryptophan residues at subsites -7 and -4 (Trp40 and Trp38 respectively) are located at the entrance and middle of the tunnel-like active site of TrCel7A, and are conserved among the GH family 7 cellobiohydrolases. Trp40 of TrCel7A is important for the recruitment of cellulose chain ends on the substrate surface, but the role of Trp38 is less clear. Comparison of the effects of W38A and W40A mutations on the binding energies of sugar units at the two subsites indicated that the contribution of Trp38 to the binding was greater than that of Trp40. In addition, the smooth gradient of binding energy was broken in W38A mutant. To clarify the importance of Trp38, the activities of TrCel7A WT and W38A towards crystalline cellulose and amorphous cellulose were compared. W38A was more active than WT towards amorphous cellulose, whereas its activity towards crystalline cellulose was only one-tenth of that of WT. To quantify the effect of mutation at subsite -4, we measured kinetic parameters of TrCel7A WT, W40A and W38A towards cello-oligosaccharides. All combinations of enzymes and substrates showed substrate inhibition, and comparison of the inhibition constants showed that the Trp38 residue increases the velocity of substrate intake (k(on) for forming productive complex) from the minus side of the subsites. These results indicate a key role of Trp38 residue in processively loading the reducing-end of cellulose chain into the catalytic tunnel.

Automated summary

The Trp38 residue in TrCel7A plays a key role in processively loading the reducing-end of cellulose chain into the catalytic tunnel, while Trp40 is important for recruiting cellulose chain ends on the substrate surface. The mutation W38A disrupts the smooth gradient of binding energy and leads to increased activity towards amorphous cellulose but decreased activity towards crystalline cellulose.