Multi-Mode Binding of Cellobiohydrolase Cel7A from Trichoderma reesei to Cellulose

Abstract

Enzymatic hydrolysis of recalcitrant polysaccharides like cellulose takes place on the solid-liquid interface. Therefore the adsorption of enzymes to the solid surface is a pre-requisite for catalysis. Here we used enzymatic activity measurements with fluorescent model-substrate 4-methyl-umbelliferyl-β-D-lactoside for sensitive monitoring of the binding of cellobiohydrolase TrCel7A from Trichoderma reesei to bacterial cellulose (BC). The binding at low nanomolar free TrCel7A concentrations was exclusively active site mediated and was consistent with Langmuir's one binding site model with K d and A max values of 2.9 nM and 126 nmol/g BC, respectively. This is the strongest binding observed with non-complexed cellulases and apparently represents the productive binding of TrCel7A to cellulose chain ends on the hydrophobic face of BC microfibril. With increasing free TrCel7A concentrations the isotherm gradually deviated from the Langmuir's one binding site model. This was caused by the increasing contribution of lower affinity binding modes that included both active site mediated binding and non-productive binding with active site free from cellulose chain. The binding of TrCel7A to BC was found to be only partially reversible. Furthermore, the isotherm was dependent on the concentration of BC with more efficient binding observed at lower BC concentrations. The phenomenon can be ascribed to the BC concentration dependent aggregation of BC microfibrils with concomitant reduction of specific surface area.

Biochemical and Structural Characterizations of Two Dictyostelium Cellobiohydrolases from the Amoebozoa Kingdom Reveal a High Level of Conservation between Distant Phylogenetic Trees of Life

“…Reaction mixtures were incubated at 30,35,40,45,50,55,60, and 65°C in a water bath. At 0, 1, 5, 10, 20, and 30 min, 150 l was removed from the reaction vial and placed in a 96-well plate containing 25 l of 1.0 M sodium carbonate.…”

“…These cellulases act from the reducing end of cellulose chains and perform multiple, processive hydrolytic events before disassociating from a cellulose chain (32). For cellulases, such as the model GH7 CBH from T. reesei (TreCel7A), this process continues until the enzyme either runs into an obstruction where the enzyme is stalled and eventually releases the substrate or the end of the cellulose chain is reached (33)(34)(35). TreCel7A exhibits the most extensively enclosed tunnel among known GH7 CBH structures, while PchCel7D displays the most open active site due to several loop deletions and residue size reductions on the tips of tunnel-enclosing loops (36).…”

The Predominant Molecular State of Bound Enzyme Determines the Strength and Type of Product Inhibition in the Hydrolysis of Recalcitrant Polysaccharides by Processive Enzymes

“…Because the number of enzyme-accessible polymer chain ends is low, the assumption of excess substrate may not hold. Because of the multimode binding and possible traffic jams on the cellulose surface, it has been suggested that the performance of Cel7A should be measured at low nanomolar enzyme concentrations (38,41). Therefore, we studied the cellobiose inhibition of Cel7A (10 nM) with 14 C-labeled BMCC (0.05-1.0 mg ml Ϫ1 ).…”

“…Whether these differences reflect methodological differences or the different substrates used is not known. Attempts to distinguish between different binding modes of bound enzymes have revealed that Cel7A is predominantly bound to cellulose through its active site (18,26,40), although the population of bound enzyme with free active site may be significant at high enzyme-to-substrate ratios (41). At the same time, enzyme attachment to cellulose via the CBM only has been reported to be the predominant state of the processive endocellulase Cel9A from the bacterium Thermobifida fusca (42), thus supporting slow complexation.…”

“…Cel7A is well known to bind cellulose predominantly through the active site (26,40), although at high enzyme-to-substrate ratios, the population of bound enzyme with its active site free may be significant (41). The noncompetitive inhibition of ChiA suggested that ChiA exists predominantly in the nonproductive E*S complex.…”