5-27: Experimental and computational investigations of cellobiohydrolase complexation behavior

In catalyzing the hydrolysis of insoluble cellulose, processive cellulase enzymes take part in complex biophysical phenomena that are poorly understood.  In this work, the influence of cellobiohydrolase surface mobility and end-of-chain association rates has been investigated with native cellulases, engineered fusions, and novel fluorescent protein reagents.  Time course product distribution plots for reconstituted mixtures of purified enzymes reveal non-equilibrium behavior that illustrates the extent to which surface mobility is rate-limiting.  The kinetic implications of surface diffusion are further explored with constructs of the Trichoderma reesei Cel7A enzyme that have been modified with CBM domains which have been demonstrated to have different surface dynamics via fluorescence photoactivation experiments.  Results and hypotheses are corroborated by a kinetic model that has been built to incorporate cellulase surface behavior that has been observed and measured experimentally.