Probing Carbohydrate Product Expulsion from a Processive Cellulase with Multiple Absolute Binding Free Energy Methods

Lintao Bu, Gregg T. Beckham, Michael R. Shirts, Mark R. Nimlos, William S. Adney, Michael E. Himmel, Michael F. Crowley
2011 Journal of Biological Chemistry  
Understanding the enzymatic mechanism that cellulases employ to degrade cellulose is critical to efforts to efficiently utilize plant biomass as a sustainable energy resource. A key component of cellulase action on cellulose is product inhibition from monosaccharide and disaccharides in the product site of cellulase tunnel. The absolute binding free energy of cellobiose and glucose to the product site of the catalytic tunnel of the Family 7 cellobiohydrolase (Cel7A) of Trichoderma reesei
more » ... ea jecorina) was calculated using two different approaches: steered molecular dynamics (SMD) simulations and alchemical free energy perturbation molecular dynamics (FEP/ MD) simulations. For the SMD approach, three methods based on Jarzynski's equality were used to construct the potential of mean force from multiple pulling trajectories. The calculated binding free energies, ؊14.4 kcal/mol using SMD and ؊11.2 kcal/mol using FEP/MD, are in good qualitative agreement. Analysis of the SMD pulling trajectories suggests that several protein residues (Arg-251, Asp-259, Asp-262, Trp-376, and Tyr-381) play key roles in cellobiose and glucose binding to the catalytic tunnel. Five mutations (R251A, D259A, D262A, W376A, and Y381A) were made computationally to measure the changes in free energy during the product expulsion process. The absolute binding free energies of cellobiose to the catalytic tunnel of these five mutants are ؊13.1, ؊6.0, ؊11.5, ؊7.5, and ؊8.8 kcal/mol, respectively. The results demonstrated that all of the mutants tested can lower the binding free energy of cellobiose, which provides potential applications in engineering the enzyme to accelerate the product expulsion process and improve the efficiency of biomass conversion.
doi:10.1074/jbc.m110.212076 pmid:21454590 pmcid:PMC3093888 fatcat:pmv7pgj73feqtmseen3dievwya