The cellulolytic ability of fungal species is important to both natural and engineered biocycling of plant matter. One essential step is the conversion of cellobiose into glucose catalyzed by beta-glucosidases. Mutagenesis studies have implicated altering the substrate binding pocket to influence the pH-activity profile of this enzyme. However, structural understanding of the pH-affected substrate binding environment is lacking. Here we conducted molecular dynamics simulations of fully hydrated TrBgl2, a beta-glucosidase of Trichoderma reesei, equilibrated at its optimal pH (pH 6) and two unfavorable pHs (pH 5 and pH 7.5). We identified structural arrangement of specific residues that facilitated substrate escape from the catalytic site at pH 5 but locked the bound substrate in an unfavorable orientation at pH 7.5. For comparative analysis, we also performed simulations of a mutated TrBgl2 with previously demonstrated improved catalysis as a function of pH. We captured the responsible conformational changes in the engineered substrate binding pocket.