Product binding varies dramatically between processive and nonprocessive cellulase enzymes

Abstract

Cellulases hydrolyze β-1,4 glycosidic linkages in cellulose, which are among the most prevalent and stable bonds in Nature. Cellulases comprise many glycoside hydrolase families and exist as processive or nonprocessive enzymes. Product inhibition negatively impacts cellulase action, but experimental measurements of product-binding constants vary significantly, and there is little consensus on the importance of this phenomenon. To provide molecular level insights into cellulase product inhibition, we examine the impact of product binding on processive and nonprocessive cellulases by calculating the binding free energy of cellobiose to the product sites of catalytic domains of processive and nonprocessive enzymes from glycoside hydrolase families 6 and 7. The results suggest that cellobiose binds to processive cellulases much more strongly than nonprocessive cellulases. We also predict that the presence of a cellodextrin bound in the reactant site of the catalytic domain, which is present during enzymatic catalysis, has no effect on product binding in nonprocessive cellulases, whereas it significantly increases product binding to processive cellulases. This difference in product binding correlates with hydrogen bonding between the substrate-side ligand and the cellobiose product in processive cellulase tunnels and the additional stabilization from the longer tunnel-forming loops. The hydrogen bonds between the substrate- and product-side ligands are disrupted by water in nonprocessive cellulase clefts, and the lack of long tunnel-forming loops results in lower affinity of the product ligand. These findings provide new insights into the large discrepancies reported for binding constants for cellulases and suggest that product inhibition will vary significantly based on the amount of productive binding for processive cellulases on cellulose.

FIGURE 1.

Structure of the CD of Cel7A (A), Cel7B (B), Cel6A (C), and Cel6B (D) with a cellodextrin chain (shown in blue stick model) at the reactant site and a cellobiose (shown in red stick model) at the product site of the CD. The cellodextrin is celloheptaose in Cel7A and Cel7B and cellotetraose in Cel6A and Cel6B. Construction of the N-linked glycans, which are shown below the glycosylated enzymes, is described in the supplemental text. The black line connecting the green and yellow spheres represents the pulling coordinate.

FIGURE 2.

PMF profiles of cellobiose expulsion from the CD of the four cellulases. PMF for Cel7A with cellodextrin is adopted from Ref. . Errors are computed by bootstrap sampling.

FIGURE 3.

A typical snapshot at 10 ns shows water molecules surrounding the carbohydrate ligand in the four cellulases examined in this study: Cel7A (A), Cel7B (B), Cel6A (C), and Cel6B (D). All water molecules within 3.5 Å of the carbohydrate substrate are shown in pink.

FIGURE 4.

Cel7A inhibition by cellobiose (modified from Ref. 9). The cellodextrin chain is shown in blue, and the cellobiose is shown in red. E, free Cel7A; ES′, precatalytic Cel7A-cellodextrin complex; ES, catalytically active Cel7A-cellodextrin complex; EI, Cel7A-inhibitor complex; ES′I, Cel7A-cellodextrin-inhibitor complex.