Product Binding Varies Dramatically between Processive and Nonprocessive Cellulase Enzymes

Abstract

Background: Experimentally measured product inhibition constants of cellobiose for cellulases vary significantly. Results: Cellodextrin bound as substrate to cellulases increases product binding to processive cellulases, whereas it does not affect nonprocessive cellulases. Conclusion:The increased binding affinity correlates with hydrogen bonds between the substrate and cellobiose in processive cellulase tunnels. Significance: The results offer an interpretation for the discrepancy in measured inhibition constants.

Loop Motions Important to Product Expulsion in the Thermobifida fusca Glycoside Hydrolase Family 6 Cellobiohydrolase from Structural and Computational Studies

“…The computed reversible thermodynamic work required to remove cellobiose from the product sites of the enzyme is the negative cellobiose binding free energy. Thus, the overall binding free energy of cellobiose calculated using all 100 SMD trajectories from TfuCel6B-cellodextrin complex and TfuCel6B-cellulose complex is Ϫ21.2 Ϯ 1.1 and Ϫ19.8 Ϯ 0.5 kcal/mol, respectively, which is consistent with the binding free energy of cellobiose to the catalytic domain of HjeCel6A, Ϫ22.4 Ϯ 0.3 kcal/mol, as characterized in our previous study (15). Specifically, for the TfuCel6B-cellodextrin complex, the calculated binding free energy is Ϫ25.9 Ϯ 1.1 kcal/mol by using 19 trajectories involving only the large opening of the exit loop and Ϫ25.4 Ϯ 0.2 kcal/mol by using 20 trajectories involving only the large opening of the bottom loop, respectively (supplemental Fig.…”

“…4, d (also referred to as the reaction coordinate), was increased with a speed of 1 Å/ns in 14 ns. We examined different pulling speeds, and 1 Å/ns was found to yield converged results similar to our previous work (15,59). The force constant is 5000 kcal/(mol ϫ Å 2 ).…”

“…Steered Molecular Dynamics (SMD) Simulations-The protocols for conducting SMD simulations on the TfuCel6B-cellodextrin complex and the TfuCel6B-cellulose complex follow our previous work on product inhibition (15,59). CHAMBER (61) was used to convert the protein structure file, coordinate file, and force field files in CHARMM format to Amber format.…”

“…Namely, endoglucanases act on amorphous regions of cellulose to create points for attachment and detachment of processive CBHs (9), which hydrolyze successive units of the cellulose chain via a multistep mechanism involving processive motion, hydrolysis, and product expulsion (8, 10 -13). ␤-Glucosidases subsequently hydrolyze soluble cellobiose to glucose to reduce CBH product inhibition (14,15).…”

Towards a molecular-level theory of carbohydrate processivity in glycoside hydrolases

“…Deglycosylation produces the 'Substrate-Product complex' wherein the cellobiose product likely sits in essentially the same position as in the 'Primed GEI'. The processive cycle is completed by product expulsion [73], resulting in a vacant +1/+2 sites ( Figure 2, top left) and a cellulose chain extending from the -1 site to beyond the -7 site and out into solution.…”