5-52: Probing carbohydrate product expulsion from a processive cellulase with multiple absolute binding free energy methods

Among the many families of cellulase enzymes contributing to cellulose deconstruction, the Family 7 cellulase (Cel7A) from Hypocrea jecorina provides most of the hydrolytic potential. A key component of Cel7A action on cellulose is product inhibition from cellobiose and glucose in the product site of Cel7A tunnel. In this work, the absolute binding free energy of cellobiose and glucose to the Cel7A catalytic tunnel was calculated using steered molecular dynamics (SMD) simulations via Jarzynski’s equality and alchemical free energy perturbation molecular dynamics (FEP/MD) simulations. The calculated binding free energies using these two different approaches are in good agreement. Several point mutations on the key binding residues identified from the SMD pulling trajectories were made computationally to study the free energy changes during the product expulsion process.