Processivity of cellobiohydrolases is limited by the substrate

Abstract

Processive cellobiohydrolases (CBHs) are the key components of fungal cellulase systems. Despite the wealth of structural data confirming the processive mode of action, little quantitative information on the processivity of CBHs is available. Here, we developed a method for measuring cellulase processivity. Sensitive fluorescence detection of enzyme-generated insoluble reducing groups on cellulose after labeling with diaminopyridine enabled quantification of the number of reducing-end exo-mode and endo-mode initiations. Both CBHs TrCel7A from Trichoderma reesei and PcCel7D from Phanerochaete chrysosporium employed reducing-end exo- and endo-mode initiation in parallel. Processivity values measured for TrCel7A and PcCel7D on cellulose hydrolysis were more than an order of magnitude lower than the values of intrinsic processivity that were found from the ratio of catalytic constant (k(cat)) and dissociation rate constant (k(off)). We propose that the length of the obstacle-free path available for a processive run on cellulose chain limits the processivity of CBHs on cellulose. TrCel7A and PcCel7D differed in their k(off) values, whereas the k(cat) values were similar. Furthermore, the k(off) values for endoglucanases (EGs) were much higher than the k(off) values for CBHs, whereas the k(cat) values for EGs and CBHs were within the same order of magnitude. These results suggest that the value of k(off) may be the primary target for the selection of cellulases.

FIGURE 1.

Principle of the measurement of cellulase processivity. A, labeling of reducing groups on cellulose via reductive amination with fluorescent DAP is shown. Elimination of water between the aldehyde group of the reducing end of the cellulose chain and the amino group of DAP results in the formation of a Schiff base intermediate (not shown), which is reduced by NaCNBH₃ to form a stable aminoalditol derivative of cellulose (DAP-cellulose). The stronger reducing agent NaBH₄ can be used to reduce aldehyde groups of cellulose to corresponding alditols, which do not give reductive amination with DAP. B, the active site tunnel of TrCel7A and PcCel7D contains 10 glucose unit binding sites (not numbered) and the cleavage site (indicated with arrowheads) is between binding sites −2 and +2. Association (k_on) results in the formation of the enzyme-substrate complex that can dissociate (k_off) or go through catalytic events (k_cat) to form cellobiose and an enzyme-substrate complex localizing one cellobiose unit further on the cellulose chain. Intrinsic processivity (P^Intr) can be found from the values of k_cat and k_off. In this study the k_off values were found on reduced cellulose (Equation 6) using the assumption that k_off is limiting for enzyme recruitment and k_cat ≫ k_off. C, release of DAP-sugar end-label (EL) in hydrolysis of DAP-cellulose reveals the number of initiations from the reducing end of cellulose ((N_init)_R-exo). D, hydrolysis of reduced cellulose and detection of enzyme-generated IRGs on residual cellulose reveals the sum of endo-initiations (N_init)_endo and (N_init)_R-exo. Apparent processivity (P^app) is given by the ratio of enzyme-released SRGs and IRGs (Equation 3). Comparison of the number of initiations measured using DAP-cellulose and reduced cellulose as substrate allows calculation of the probability for endo-initiation (P_Endo, Equation 4).

FIGURE 2.

Comparative hydrolysis of DAP-cellulose and reduced cellulose reveals the use of endo-mode initiation by cellobiohydrolases TrCel7A and PcCel7D. Experiments were performed in 50 mm sodium acetate, pH 5.0, at 30 °C. Cellulose concentration was 1.0 g liter⁻¹, and enzyme was 20 nm TrCel7A (□ and ■) or PcCel7D (△ and ▲). Open symbols (□ and △) refer to DAP-cellulose, and filled symbols (■ and ▲) refer to reduced cellulose. Error bars are from three independent measurements. A, release of SRGs in hydrolysis of DAP-BC and rBC is shown. B, release of EL in hydrolysis of DAP-BC and formation of IRGs in hydrolysis of rBC is shown. C, release of SRGs in hydrolysis of DAP-AC and reduced rAC is shown. D, release of EL in hydrolysis of DAP-AC and formation of IRGs in hydrolysis of rAC is shown.

FIGURE 3.

Apparent processivity of cellulases on bacterial cellulose and amorphous cellulose. Experiments were performed in 50 mm sodium acetate, pH 5.0, at 30 °C. Reduced cellulose was used as a substrate. SRGs were measured using the BCA method, and formation of IRGs was measured using fluorescence labeling. The amount of RG_tot was found from the sum of SRGs and IRGs. Solid lines are from the linear regression, and the slope of the line equals the apparent processivity (P^app, Equation 3). A, reduced bacterial cellulose was 1.0 g liter⁻¹, and enzyme was 20 nm TrCel7A (□) or PcCel7D (□). Error bars are from three independent measurements. B, in the case of cellobiohydrolases TrCel7A (□) and PcCel7D (△), the concentration of reduced amorphous cellulose (rAC) was 1.0 g liter⁻¹, and enzyme varied between 5 and 100 nm. In the case of endoglucanases TrCel5A (♢) and TrCel12A (×), the concentration of enzyme was 20 nm, and rAC was 3.0 and 5.0 g liter⁻¹, respectively. Error bars are from three independent measurements.

FIGURE 4.

Rapid slowdown in hydrolysis rates is characteristic for cellobiohydrolases. Experiments were performed in 50 mm sodium acetate, pH 5.0, at 30 °C. Reduced amorphous cellulose (2.0 g liter⁻¹) was used as substrate, and the enzyme was 0.1 μm cellobiohydrolase TrCel7A (□), PcCel7D (△), or endoglucanase TrCel5A (♢). The amount of RG_Tot was measured using the BCA method. The inset shows the initial burst of RG_Tot observed with CBHs. Error bars are from three independent measurements.