The impact of active site protonation on substrate ring conformation in Melanocarpus albomyces cellobiohydrolase Cel7B

Abstract

The ability to utilize biomass as a feedstock for liquid fuel and value-added chemicals is dependent on the efficient and economic utilization of lignin, hemicellulose, and cellulose. In current bioreactors, cellulases are used to convert crystalline and amorphous cellulose to smaller oligomers and eventually glucose by means of cellulase enzymes. A critical component of the enzyme catalyzed hydrolysis reaction is the degree to which the enzyme can facilitate substrate ring deformation from the chair to a more catalytically active conformation (e.g. skewed boat) at the −1 subsite. Presented here is an evaluation of the impact of the protonation state for critical active site residues (i.e. Glu212, Asp214, Glu217, and His228) in Melanocarpus albomyces (Ma) Cellobiohydrolase Cel7B on the substrate's orientation and ring conformation. It is found that the protonation state of the active site can disrupt the intra-enzyme hydrogen bonding network and enhance the sampling of various ring puckering conformations for the substrate ring at the +1 and −1 subsites. In particular it is observed that the protonation state of Asp214 dictates the accessibility of the glycosidic bond to the catalytic acid/base Glu217 by influencing the φ/ψ dihedral angles and the puckering of the ring structure. The protonation–orientation–conformation analysis has revealed an active site that primarily utilizes two highly coupled protonation schemes; one protonation scheme to orient the substrate and generate catalytically favorable substrate geometries and ring puckering conformations and another protonation scheme to hydrolyze the glycosidic bond. In addition to identifying how enzymes utilize protonation state to manipulate substrate geometry, this study identifies possible directions for improving catalytic activity through protein engineering.