Endo-exo synergism in cellulose hydrolysis revisited

Abstract

Synergistic cooperation of different enzymes is a prerequisite for efficient degradation of cellulose. The conventional mechanistic interpretation of the synergism between randomly acting endoglucanases (EGs) and chain end-specific processive cellobiohydrolases (CBHs) is that EG-generated new chain ends on cellulose surface serve as starting points for CBHs. Here we studied the hydrolysis of bacterial cellulose (BC) by CBH TrCel7A and EG TrCel5A from Trichoderma reesei under both single-turnover and "steady state" conditions. Unaccountable by conventional interpretation, the presence of EG increased the rate constant of TrCel7A-catalyzed hydrolysis of BC in steady state. At optimal enzyme/substrate ratios, the "steady state" rate of synergistic hydrolysis became limited by the velocity of processive movement of TrCel7A on BC. A processivity value of 66 ± 7 cellobiose units measured for TrCel7A on (14)C-labeled BC was close to the leveling off degree of polymerization of BC, suggesting that TrCel7A cannot pass through the amorphous regions on BC and stalls. We propose a mechanism of endo-exo synergism whereby the degradation of amorphous regions by EG avoids the stalling of TrCel7A and leads to its accelerated recruitment. Hydrolysis of pretreated wheat straw suggested that this mechanism of synergism is operative also in the degradation of lignocellulose. Although both mechanisms of synergism are used in parallel, the contribution of conventional mechanism is significant only at high enzyme/substrate ratios.

FIGURE 1.

Hydrolysis of ¹⁴C-BC by TrCel7A under single-turnover conditions. A, ¹⁴C-BC (0.5 mg ml⁻¹) was incubated with 1.0 μm TrCel7A. An AC trap was added after 10 s (□), 30 s (♢), or 60 s (△) of hydrolysis, and the release of ¹⁴CB in time was followed. B, [TrCel7A]_OA was measured from the inhibition of the hydrolysis of MUL by BC (0.5 mg ml⁻¹). [TrCel7A] was 0.1 μm (○), 0.25 μm (△), 0.5 μm (♢), or 1.0 μm (□). Solid lines are from the non-linear regression according to Equation 2. C, data from A divided by the corresponding concentration of TrCel7A at the moment of the AC trap addition ([ES]_trap). Values of [ES]_trap were taken from B using an assumption that [ES]_trap = [TrCel7A]_OA. D, average [¹⁴CB]/[ES]_trap ratios over experiments with different TrCel7A concentrations and different times of trap addition. Shown is non-linear regression according to the single exponent (Equation 3; dotted line) or to the sum of two exponents (Equation 4; solid line). The dashed line and the dashed and dotted line represent the fast and slow components of Equation 4, respectively. Error bars show S.D. and are from at least three independent measurements.

FIGURE 2.

Synergism in hydrolysis of ¹⁴C-BC by TrCel7A and EG TrCel5A under single-turnover conditions. ♢, TrCel7A; ♦, TrCel7A + EG; △, CD_TrCel7A; ▴, CD_TrCel7A + EG. Concentration of TrCel7A or CD_TrCel7A was 1.0 μm. If present, the concentration of EG (TrCel5A) was 0.1 μm. A, release of ¹⁴CB in hydrolysis of ¹⁴C-BC (0.5 mg ml⁻¹). AC trap was added after 10 s of hydrolysis. B, concentration of TrCel7A with the active site occupied by the cellulose chain ([TrCel7A]_OA) was measured from the inhibition of the hydrolysis of MUL by BC (0.5 mg ml⁻¹). Solid lines are from the non-linear regression according to Equation 2. Error bars show S.D. and are from at least three independent measurements.

FIGURE 3.

Synergism in hydrolysis of BC by TrCel7A and EG TrCel5A. TrCel7A (♢), TrCel7A + EG (♦), CD_TrCel7A (△), CD_TrCel7A + EG (▴). Concentration of TrCel7A or CD_TrCel7A was 0.5 μm, and that of β-glucosidase was 0.85 μm. If present, the concentration of EG was 0.1 μm. Error bars show S.D. and are from three independent measurements. In A–C, concentration of BC was 0.5 mg ml⁻¹. A, glucose formation. B, concentration of TrCel7A with the active site occupied by the cellulose chain ([TrCel7A]_OA) was measured from the inhibition of the hydrolysis of MUL by BC (0.5 mg ml⁻¹). C, values of the observed rate constant (k_obs) were found from the rates of cellobiose formation (recalculated from the rates of glucose formation) and values of [TrCel7A]_OA according to Equation 6. D–F, data points were taken after 2, 5, and 10 min of hydrolysis. D, glucose formation after 10 min of hydrolysis. E, average values over all time points are plotted. Because of systematic variation in time, only 10 min data point values are plotted for the series with CD_TrCel7A + EG. F, average k_obs values over all time points are plotted. DSE values at different BC concentrations are listed in Table 2.

FIGURE 4.

Synergism in hydrolysis of lignocellulose by TrCel7A and EG TrCel5A. ♢, TrCel7A; ♦, TrCel7A + EG; △, CD_TrCel7A; ▴, CD_TrCel7A + EG. Concentration of TrCel7A or CD_TrCel7A was 2.5 μm, and that of β-glucosidase was 0.85 μm. If present, the concentration of EG was 0.25 μm. Hydrolysis time was 30 min. Error bars show S.D. and are from three independent measurements. A, glucose formation. B, concentration of TrCel7A with the active site occupied by the cellulose chain ([TrCel7A]_OA) was measured from the inhibition of the hydrolysis of pNPL by lignocellulose. C, values of observed rate constant (k_obs) were found from the rates of cellobiose formation (recalculated from the rates of glucose formation) and values of [TrCel7A]_OA according to Equation 6. DSE values at different lignocellulose concentrations are listed in supplemental Table S3.

FIGURE 5.

Cellobiose inhibition of TrCel7A on ¹⁴C-BC under single-turnover conditions. Error bars show S.D. and are from three independent measurements. A, release of ¹⁴CB in the presence of added cellobiose at 0 mm (♢), 0.5 mm (△), 2.0 mm (○), 5.0 mm (×), or 20 mm (□). Concentration of ¹⁴C-BC and TrCel7A was 0.5 mg ml⁻¹ and 1.0 μm, respectively. Series without added cellobiose were provided with 0.125 μm β-glucosidase. An AC trap was added after 30 s of hydrolysis. B and C, effect of added cellobiose on parameters of single-turnover kinetics, [¹⁴CB]_max and k. Values of parameters (listed in supplemental Table S4) were found by non-linear regression of the hydrolysis data in A (for TrCel7A) or supplemental Fig. S2 (for TrCel7A + EG) according to Equation 1. Subscripts CB and CB = 0 refer to the parameters measured in the presence and absence of cellobiose, respectively. ♢, TrCel7A; ♦, TrCel7A + EG.

FIGURE 6.

Cellobiose inhibition of TrCel7A on ¹⁴C-BC in “steady state.” A and B, release of ¹⁴CB in hydrolysis of ¹⁴C-BC by individual TrCel7A (A) or TrCel7A + EG (B). The concentration of ¹⁴C-BC and TrCel7A was 0.25 mg ml⁻¹ and 0.25 μm, respectively. If present, the concentration of EG (TrCel5A) was 0.025 μm. Series without added cellobiose were provided with 0.125 μm β-glucosidase (♢). Concentration of added cellobiose was 0 mm (♢), 0 mm without β-glucosidase (♦), 0.2 mm (*), 0.5 mm (△), 1.0 mm (+), 2.0 mm (○), 5.0 mm (×), or 10 mm (□). C and D, hydrolysis data were rearranged to obtain the ratio of ([¹⁴CB]_CB/[¹⁴CB]_{CB = 0}) for each time point. Subscripts CB and CB = 0 refer to the measurements in the presence and absence of cellobiose, respectively. Plotted are average values over all time points (except TrCel7A + EG) with corresponding error bars (S.D.). Solid lines are from the non-linear regression according to supplemental Equation S1 (individual enzymes) or Equation 9 (synergistic mixtures). C, TrCel7A (♢) and TrCel7A + EG (♦). D, CD_TrCel7A (△) and CD_TrCel7A + EG (▴).

FIGURE 7.

Hydrolysis of cellulose by processive CBH (A and B) and possible mechanism of synergism with EG (C). A, processive cycle of CBH consists of at least six putative steps as follows (65). a, CBM-mediated binding to the cellulose surface; b, finding and recognition of cellulose chain end; c, threading of cellulose chain into active site tunnel and formation of productive enzyme-substrate complex; d, hydrolysis of glycosidic bond; e, expulsion of product CB; f, threading another cellobiose unit to reform productive complex. Steps d–f are repeated until enzyme happens to dissociate or stops behind an obstacle (55). Transit times were found from the values of rate constants for the corresponding step(s). B, hydrolysis of cellulose by individual CBH. CBH cannot pass through the amorphous regions (wavy lines) and stalls. The length of the crystalline regions between amorphous parts defines the length of obstacle-free path (n_free), which also limits the apparent processivity (P_app) of CBH. The steady state rate of cellobiose formation is governed by the slow dissociation (k_off value was taken from Ref. 57) of stalled CBH. C, synergistic hydrolysis of cellulose. EG accelerates the recruitment of CBH by degrading amorphous regions. P_app of CBH is determined by the DP of EG-fragmented cellulose surface (DP_surface), and the steady state rate of cellobiose formation approaches the limit set by the velocity of processive movement of CBH. Transit times correspond to the steps taken by the CBH depicted to the left of the cellulose. Conventional mechanism of endo-exo synergism is depicted to the right of the cellulose.