Substrate binding in the processive cellulase Cel7A: Transition state of complexation and roles of conserved tryptophan residues

Abstract

Cellobiohydrolases effectively degrade cellulose and are of biotechnological interest because they can convert lignocellulosic biomass to fermentable sugars. Here, we implemented a fluorescence-based method for real-time measurements of complexation and decomplexation of the processive cellulase Cel7A and its insoluble substrate, cellulose. The method enabled detailed kinetic and thermodynamic analyses of ligand binding in a heterogeneous system. We studied WT Cel7A and several variants in which one or two of four highly conserved Trp residues in the binding tunnel had been replaced with Ala. WT Cel7A had on/off-rate constants of 1 × 105m−1 s−1 and 5 × 10−3 s−1, respectively, reflecting the slow dynamics of a solid, polymeric ligand. Especially the off-rate constant was many orders of magnitude lower than typical values for small, soluble ligands. Binding rate and strength both were typically lower for the Trp variants, but effects of the substitutions were moderate and sometimes negligible. Hence, we propose that lowering the activation barrier for complexation is not a major driving force for the high conservation of the Trp residues. Using so-called Φ-factor analysis, we analyzed the kinetic and thermodynamic results for the variants. The results of this analysis suggested a transition state for complexation and decomplexation in which the reducing end of the ligand is close to the tunnel entrance (near Trp-40), whereas the rest of the binding tunnel is empty. We propose that this structure defines the highest free-energy barrier of the overall catalytic cycle and hence governs the turnover rate of this industrially important enzyme.

Enzymatic processing of lignocellulosic biomass: principles, recent advances and perspectives

“…Despite much progress in the enzyme area, further improvements still seem possible. For example, it is still not fully clear how processive cellulases work and how the interplay of these essential but rather slow enzymes with other enzymes could be optimized [57,169,302,306,362]. Recent insights concerning the role of H 2 O 2 and enzyme inactivation suggest that so far, we have not harnessed the full potential of LPMOs.…”

A comparative biochemical investigation of the impeding effect of C1-oxidizing LPMOs on cellobiohydrolases

“…The effect of substrate oxidation on the activity of the three cellobiohydrolases was further investigated by a fluorescence-based method that detects ligand–enzyme complexation ( 35 ). The substrate-binding tunnel of cellobiohydrolases is decorated with tryptophan residues that interact with the cellulose chain ( 36 ).…”

“…S4 ) in an enzyme–substrate complex as a function of time after injection of substrate. The fraction of enzymes in a substrate complex, Φ threaded, was calculated based on the increment in the fluorescence signal at saturation (where E 0 ∼ [ES]), F max as, where F x is the increment in signal in an experiment with a given cellulose load ( 35 ).

Figure 4 Real-time fluorescence data for the complexation of phosphoric acid swollen cellulose (PASC) and oxidized PASC.

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“…The method has a dead time of a few hundred milliseconds ( 35 ), and this interval was thus ignored in the data analysis. The method allows for simple determination of the on-rate, v on , as the initial slope of the fluorescence signal.…”

Nanoscale Dynamics of Cellulase TrCel7A Digesting Cellulose

“…The exoglucanase Cel7A from Trichoderma reesei (teleomorph Hypocrea jecorina) is a model cellulase enzyme that degrades the glucan chains of cellulose from their reducing ends. Its mechanism of action has been investigated using traditional biochemical methods (6)(7)(8)(9), AFM (10,11), single-molecule imaging (12)(13)(14), and optical tweezers (15), and these results have contributed to quantitative kinetic models of Cel7A activity (1, 2,16,17). Cel7A moves processively at a few nm/s, can operate against an external load of 20 pN, and appears to get stuck in "traffic jams" at high enzyme concentrations (11,15).…”