Molecular dynamics studies of the catalytic mechanism of a Family 3 β-glucosidase
β-glucosidases play an important role in the enzymatic hydrolysis of cellulose by relieving endoglucanase and cellobiohydrolase product inhibition through hydrolysis of cellobiose to glucose. Among those used in commercial enzyme cocktails is Cel3A from Hypocrea jecorina (HjCel3A), which was found to enhance the conversion of various cellulosic substrates by nearly 10%. However, the catalytic activity of HjCel3A and other β-glucosidases is significantly reduced at high cellobiose or glucose concentration owing to their susceptibility to transglycosylation, a competing pathway to hydrolysis, wherein glucose or another cellobiose molecule, instead of water, attacks the glycosyl-enzyme intermediate to form a di- or trisaccharide. Studies on enzymes that catalyze a similar reaction suggest that the hydrolysis/transglycosylation ratio may be influenced by the pKa of active site residues. Molecular dynamics (MD) will be employed to determine the mechanisms of the initial glycosylation step and the subsequent hydrolysis or transglycosylation, as well as to understand the pH dependence of the catalytic activity of HjCel3A. The minimum energy pathway (MEP) of each reaction step will be determined using the adiabatic mapping approach, wherein potential energy surfaces (PES) along the chosen reaction coordinate(s) are generated. The free energy profile (FEP) will then be calculated using quantum mechanics/molecular mechanics (QM/MM) umbrella sampling. The chemically active region will be treated by the self-consistent-charge density-functional tight-binding (SCC-DFTB) method while the rest of the protein and solvent molecules will be described using the CHARMM force field. For each reaction step, the pKa of active site residues and the impact of factors such as charge coupling and hydrogen bonding will be studied by performing MD simulations at constant pH, which allows the ionizable residues to continuously switch between the protonated and deprotonated states through the introduction of an alchemical titration coordinate. This method will be coupled with pH-based replica exchange (pH-REX) to enhance the sampling of protonation states and accelerate the convergence of calculated pKa values. The research findings are expected to provide a better understanding of the factors that currently limit the catalytic activity of HjCel3A and other β-glucosidases, thus enabling the protein engineering of highly active variants.