Toward A Structural Basis For The Ph-Dependent Activity Of Beta-Glucosidases: Computational Evidence Of Conformational And Substrate-Binding Dynamics

Abstract

The complete degradation of cellulose to glucose is essential to carbon turnover in terrestrial ecosystems and to engineered biofuel production. A rate-limiting step in this pathway is catalyzed by beta-glucosidase (BGs) enzymes, which convert cellulobiose into two glucose molecules. The activity of these enzymes has been shown to vary with solution pH but the influence of pH-induced changes on the enzyme conformation required for catalytic action on the substrate is not well understood. We applied molecular dynamics simulations to investigate both conformational and substrate binding dynamics in two well-characterized BGs of bacterial (Clostridium cellulovarans) and fungal (Trichoderma reesi) origins as a function of pH. The enzymes were simulated in an explicit solvated environment, with NaCl as electrolytes, at their prominent ionization states obtained at pH 5, 6, 7, and 7.5. Our findings indicated that pH-dependent changes in the ionization states of non-catalytic residues localized outside of the immediate active site led to pH-dependent disruption of favorable H-bonding interactions with catalytic residues required to initiate catalysis on the substrate. We also identified specific non-catalytic residues that are involved in stabilizing the substrate at the optimal pH for enzyme activity. The simulations further revealed the dynamics of water-bridging interactions both outside and inside the substrate binding cleft during structural changes in the enzyme-substrate complex. These new insights provided by our findings contribute to a structural basis for the pH-dependent substrate binding specificity in BGs.