Carbohydrate–Protein Interactions That Drive Processive Polysaccharide Translocation in Enzymes Revealed from a Computational Study of Cellobiohydrolase Processivity

Abstract

Translocation of carbohydrate polymers through protein tunnels and clefts is a ubiquitous biochemical phenomenon in proteins such as polysaccharide synthases, glycoside hydrolases, and carbohydrate-binding modules. Although static snapshots of carbohydrate polymer binding in proteins have long been studied via crystallography and spectroscopy, the molecular details of polysaccharide chain processivity have not been elucidated. Here, we employ simulation to examine how a cellulose chain translocates by a disaccharide unit during the processive cycle of a glycoside hydrolase family 7 cellobiohydrolase. Our results demonstrate that these biologically and industrially important enzymes employ a two-step mechanism for chain threading to form a Michaelis complex and that the free energy barrier to chain threading is significantly lower than the hydrolysis barrier. Taken with previous studies, our findings suggest that the rate-limiting step in enzymatic cellulose degradation is the glycosylation reaction, not chain processivity. Based on the simulations, we find that strong electrostatic interactions with polar residues that are conserved in GH7 cellobiohydrolases, but not in GH7 endoglucanases, at the leading glucosyl ring provide the thermodynamic driving force for polysaccharide chain translocation. Also, we consider the role of aromatic-carbohydrate interactions, which are widespread in carbohydrate-active enzymes and have long been associated with processivity. Our analysis suggests that the primary role for these aromatic residues is to provide tunnel shape and guide the carbohydrate chain to the active site. More broadly, this work elucidates the role of common protein motifs found in carbohydrate-active enzymes that synthesize or depolymerize polysaccharides by chain translocation mechanisms coupled to catalysis.

Slow Off-rates and Strong Product Binding Are Required for Processivity and Efficient Degradation of Recalcitrant Chitin by Family 18 Chitinases

“…The encounter of an obstacle stops the processive run and leaves the enzyme in the non-productive complex with the polymer chain end in the substrate-binding site Ϫ1 (for enzymes moving toward the non-reducing end of the substrate). In the case of processive enzymes, the strong binding in the product-binding sites ϩ1 and ϩ2 creates a cumulative force to ensure directional progression of the enzyme from the nonproductive complex (polymer chain end in Ϫ1) to the productive complex (polymer chain end in ϩ2) (3,19,64,65). Therefore, it is tempting to speculate that strong binding of the enzyme in the non-productive complex dictated by low k off values helps to hold the enzyme behind obstacles in a stand-by position, whereas the force directed to fill the product-binding sites generates a "pushing potential" that, if strong enough, can remove or displace obstacles (Fig.…”

Nanoscale Engineering of Designer Cellulosomes

“…The latter has been recently elucidated in the case of cellobiohydrolases by means of classical and QM/MM MD simulations, combined with umbrella sampling and transition path sampling techniques. [199,200] The simulations elucidated the processive two-step catalysis starting at the end of a cellulose chain. The first step involves glycosylation of the cellulose chain by proton transfer via a glutamate residue (Glu217).…”

“…[201] Simulations quantify the thermodynamics of the entire process with free energy barriers associated with each step, showing that neither the activation step (ΔG = 2.9 kcal mol −1 ) nor cellobiose release (ΔG = 11.8 kcal mol −1 ) are the limiting step but that glycosylation is (ΔG = 15.5 kcal mol −1 ). [200] Such insights are key to speeding up rational design of nanobiomaterials; structures and reaction schemes which look good on paper are often misleading, meaning rigorous, generally multi-scale, modeling is required to accelerate progress in materials discovery, optimization and re-engineering. [202] 6.…”

The Predominant Molecular State of Bound Enzyme Determines the Strength and Type of Product Inhibition in the Hydrolysis of Recalcitrant Polysaccharides by Processive Enzymes

“…The CD of ChiA contains 4 substrate (Ϫ4 to Ϫ1)-and 3 product (ϩ1 to ϩ3)-binding sites (7)(8)(9), although in ChiA, the substratebinding sites extend to the CBM (10), resulting in a total of 13 substrate-binding sites (8). To ensure the directional progression of the enzyme along the polymer chain, the active sites of processive enzymes possess a binding energy gradient, with the strongest binding occurring at product-binding sites (11)(12)(13). Thus, strong product binding, and hence strong product inhibition, appears to be a price of processivity (14).…”