The Tryptophan Residue at the Active Site Tunnel Entrance of Trichoderma reesei Cellobiohydrolase Cel7A Is Important for Initiation of Degradation of Crystalline Cellulose*

Background: Mutation of Trp-40 in the Cel7A cellobiohydrolase from Trichoderma reesei (TrCel7A) causes a loss of crystalline cellulose-degrading ability. Results: Mutant W40A showed reduced specific activity for crystalline cellulose and diffused the cellulose chain from the entrance of the active site tunnel. Conclusion: Trp-40 is essential for chain end loading to initiate processive hydrolysis of TrCel7A. Significance: The mechanisms of crystalline polysaccharide degradation are clarified. The glycoside hydrolase family 7 cellobiohydrolase Cel7A from Trichoderma reesei is one of the best studied cellulases with the ability to degrade highly crystalline cellulose. The catalytic domain and the cellulose-binding domain (CBD) are both necessary for full activity on crystalline substrates. Our previous high-speed atomic force microscopy studies showed that mutation of Trp-40 at the entrance of the catalytic tunnel drastically decreases the ability to degrade crystalline cellulose. Here, we examined the activities of the WT enzyme and mutant W40A (with and without the CBD) for various substrates. Evaluation and comparison of the specific activities of the enzymes (WT, W40A, and the corresponding catalytic subunits (WTcat and W40Acat)) adsorbed on crystalline cellulose indicated that Trp-40 is involved in recruiting individual substrate chains into the active site tunnel to initiate processive hydrolysis. This was supported by molecular dynamics simulation study, i.e. the reducing end glucose unit was effectively loaded into the active site of WTcat, but not into that of W40Acat, when the simulation was started from subsite −7. However, when similar simulations were carried out starting from subsite −5, both enzymes held the substrate for 50 ns, indicating that the major difference between WTcat and W40Acat is the length of the free chain end of the substrate required to allow initiation of processive movements; this also reflects the difference between crystalline and amorphous celluloses. The CBD is important for enhancing the enzyme population on crystalline substrate, but it also decreases the specific activity of the adsorbed enzyme, possibly by attaching the enzyme to non-optimal places on the cellulose surface and/or hindering processive hydrolysis.

The glycoside hydrolase family 7 cellobiohydrolase Cel7A from Trichoderma reesei is one of the best studied cellulases with the ability to degrade highly crystalline cellulose. The catalytic domain and the cellulose-binding domain (CBD) are both necessary for full activity on crystalline substrates. Our previous high-speed atomic force microscopy studies showed that mutation of Trp-40 at the entrance of the catalytic tunnel drastically decreases the ability to degrade crystalline cellulose. Here, we examined the activities of the WT enzyme and mutant W40A (with and without the CBD) for various substrates. Evaluation and comparison of the specific activities of the enzymes (WT, W40A, and the corresponding catalytic subunits (WT cat and W40A cat )) adsorbed on crystalline cellulose indicated that Trp-40 is involved in recruiting individual substrate chains into the active site tunnel to initiate processive hydrolysis. This was supported by molecular dynamics simulation study, i.e. the reducing end glucose unit was effectively loaded into the active site of WT cat , but not into that of W40A cat , when the simulation was started from subsite ؊7. However, when similar simulations were carried out starting from subsite ؊5, both enzymes held the substrate for 50 ns, indicating that the major difference between WT cat and W40A cat is the length of the free chain end of the substrate required to allow initiation of processive move-ments; this also reflects the difference between crystalline and amorphous celluloses. The CBD is important for enhancing the enzyme population on crystalline substrate, but it also decreases the specific activity of the adsorbed enzyme, possibly by attaching the enzyme to non-optimal places on the cellulose surface and/or hindering processive hydrolysis.
Cellulose is a linear polymer of ␤1,4-linked D-glucose and is the main component of the plant cell wall. The major cellulase produced by the cellulolytic ascomycete Trichoderma reesei is the glycoside hydrolase (GH) 4 family 7 cellobiohydrolase (TrCel7A). TrCel7A has a two-domain structure composed of a catalytic domain and a CBM1 (carbohydrate-binding module 1) family cellulose-binding domain (CBD) connected by a glycosylated linker peptide (1). The three-dimensional x-ray structure of the TrCel7A catalytic domain with cello-oligosaccharides has been determined, revealing a long active site tunnel formed by loops that extend from the central ␤-sandwich fold and that are stabilized by 10 disulfide bridges (2). This tunnel contains nine glucosyl-binding sites (Ϫ7 to ϩ2), and the catalytic site (Ϫ1/ϩ1) is located at the far end, leading to production of cellobiose from the reducing end of the cellulose chain. The tunnel is aligned by hydrogen bond-forming residues, and in addition, four tryptophan residues provide hydrophobic stacking interactions for the glucosyl units at the tunnel entrance (Trp-40), at the center of the tunnel (Trp-38), and around the catalytic sites (Trp-367 and Trp-376), as shown in Fig. 1. It is believed that the tight holding of the substrate and the asymmetry of the active site tunnel favor effective and continuous hydrolysis of crystalline cellulose in a processive mode (2,3).
The processivity of cellobiohydrolases has been estimated by measurement of the ratio of released cellobiose to the sum of released glucose and cellotriose (4 -6), by product analysis of a mixture of cellulose and p-nitrophenyl lactoside (7), or by the use of labeled cellulose substrates (8,9). Recently, we demonstrated the sliding movement of single TrCel7A molecules on a highly crystalline cellulose surface by real-time monitoring with a high-speed atomic force microscope (10 -12). We showed that an inactive mutant did not slide on a cellulose surface and also that the CBD was unnecessary for the sliding movement. These studies showed that the sliding movement reflected processive hydrolysis by the TrCel7A catalytic domain. Moreover, the atomic force microscopy results demonstrated that the TrCel7A W40A mutant could not slide on highly crystalline cellulose, even though it was active toward non-crystalline substrates. We interpreted these results as showing that not only catalysis but also chain end recognition is important for effective sliding on and degradation of crystalline cellulose.
In this study, we prepared four samples of TrCel7A, i.e. WT, W40A, and the corresponding catalytic subunits (WT cat and W40A cat ) without the CBD. We then characterized their hydrolytic activity and binding properties for defined crystalline and amorphous cellulose substrates to examine the contributions of Trp-40 and the CBD to cellulose degradation. We also carried out molecular dynamics simulations of WT cat and W40A cat to elucidate the mechanism through which Trp-40 promotes processive hydrolysis of cellulose chains.

EXPERIMENTAL PROCEDURES
Construction of Mutant-encoding Plasmid and Transformation of T. reesei-Escherichia coli strain DH5␣ (Promega) was used as the cloning host for DNA construction. The pEM-F5 plasmid containing the cel7A gene (cDNA) under the control of its own promoter was used as an expression vector (13), and the T. reesei ALKO 3413 strain (Roal Ltd.) lacking the genes coding for endogenous Cel7A and Cel7B was used as the host for production of the mutant enzyme. For selection of T. reesei transformants, the hygromycin selection plasmid pRLMex30 was used (14). The mutant expression plasmid was constructed by overlap extension PCR, and the DNA sequence of the whole mutated area was confirmed by DNA sequencing. Transformation and choice of the most productive transformant were performed basically as described previously (15).
Isolation of TrCel7A and Mutant Enzymes-The production and purification of intact TrCel7A and the catalytic module of TrCel7A were performed as described previously (16). The TrCel7A W40A mutant was produced in a Chemap LF 20 fermenter (working volume of 15 liters). The cultivation medium used contained 60 g/liter whey powder, 30 g/liter spent grain, 5.0 g/liter KH 2 PO 4 , and 5 g/liter (NH 4 ) 2 SO 4 . Inocula (spore suspensions in 50% glycerol at Ϫ80°C) were grown on the same medium with only 20 g/liter carbon source and 10 g/liter spent grain, buffered with 15 g/liter KH 2 PO 4 , in two stages of 4ϫ 50 ml (3 days, 200 rpm, 28°C) and 5ϫ 200 ml (10% (v/v) transfer, 2 days, 200 rpm, 28°C). The fermentation lasted for 192 h, and the other cultivation conditions were as follows: temperature, 28°C; pH 4 -5 (lower limit controlled by the addition of NH 4 OH and upper limit controlled by the addition of H 3 PO 4 ); dissolved oxygen, Ͼ30% (agitation at 400 -800 rpm); and aeration, 5 liters/min. Foaming was controlled by the automatic addition of Struktol J 633 polyoleate antifoaming agent (SchillϩSeilacher GmbH). After fermentation, the biomass was separated by centrifugation at 4000 ϫ g for 30 min at 4°C. The culture filtrate contained at least 1 g/liter W40A protein as judged by Western blot analysis (see below).
Purification of the W40A mutants was started by adjusting the clarified Trichoderma culture filtrate (300 ml) in a Bio-Gel P-6 gel filtration column to 50 mM sodium acetate buffer (pH 5.6). The sample (containing both intact W40A and its catalytic domain (W40A cat )) was then applied to a DEAE-Sepharose FF column equilibrated with 50 mM sodium acetate (pH 5.6). The enzyme was eluted with a linear gradient of 0 -0.5 M NaCl in the equilibration buffer. The fractions containing the highest activity for 4-methylumbelliferyl ␤-D-lactoside (MULac) were pooled and adjusted to pH 6.0. Ammonium sulfate was added to 0.65 M, and the sample was loaded onto a phenyl-Sepharose FF column equilibrated with 50 mM potassium phosphate (pH 6.0) containing 0.65 M ammonium sulfate. The unadsorbed proteins, including the W40A catalytic module (W40A cat ), were eluted with the equilibration buffer, and the adsorbed W40A protein was eluted with a decreasing linear gradient to 50 mM potassium phosphate buffer (pH 6.0). Analysis of the fractions was performed using MULac as a substrate. SDS-PAGE and Western blotting were also employed as described previously (15). The purest fractions containing intact two-domain W40A and W40A cat were separately combined into two pools. The buffer of both pools was exchanged to 50 mM sodium acetate buffer (pH 5.0) in a Bio-Gel P-6 column equilibrated with the same buffer. The fractions containing the highest protein concentration were pooled, concentrated, and used for the biochemical characterization described below. The results from SDS-PAGE of the purified enzymes are shown in supplemental Fig. S1. The contaminating activities for 4-methylum- Ϫ7 is shown in red, and Glu-212, which acts as the catalytic nucleophile between subsites Ϫ1 and ϩ1 and was used as a reference position in the molecular dynamics simulations, is shown in magenta.
The protein concentration of TrCel7A preparations was measured in terms of UV absorption at 280 nm. For the WT enzyme, ⑀ ϭ 83,000 M Ϫ1 cm Ϫ1 was used, and for WT cat , ⑀ ϭ 80,000 M Ϫ1 cm Ϫ1 (based on quantitative amino acid analysis) was used. 5 For W40A and W40A cat , theoretical ⑀ values of 77,500 and 74,500 M Ϫ1 cm Ϫ1 were used, respectively; these were calculated from the measured values for the wild-type proteins by subtracting ⑀ ϭ 5,500 M Ϫ1 cm Ϫ1 for a tryptophan residue (18).
Degradation of MULac-Enzyme activities for MULac were determined by monitoring the amount of released methylumbelliferone at various substrate concentrations (0 -3.0 mM) in 50 mM sodium acetate buffer (pH 5.0) at 27°C. The enzyme concentrations used were 0.3 M for WT, WT cat , W40A, and W40A cat . The enzyme reactions were stopped by the addition of 0.5 M Na 2 CO 3 , and liberation of methylumbelliferone was measured as described by Voutilainen et al. (19). The data obtained were fitted to the Michaelis-Menten equation. To calculate the kinetic parameters for substrate inhibition, the following equation was used for curve fitting: where E 0 is the enzyme concentration, S is the substrate concentration, K m is the Michaelis constant, K m2 is the substrate inhibition constant, and k cat is the catalytic constant. All kinetic parameters were calculated using DeltaGraph 5.5.5 (Red Rock Software) or KaleidaGraph 3.6.4 (Synergy Software).
Cellulose Preparation-Highly crystalline cellulose (HCC) was prepared from green alga Cladophora spp. as described previously (20). Phosphoric acid-swollen cellulose (PASC) was prepared from Avicel (Funakoshi Corp.) as follows. Avicel was mixed with 85% (w/w) phosphoric acid, and a completely clear solution was obtained by smashing with a glass stick. After overnight incubation at 4°C, cellulose was regenerated in water, and a suspension was prepared in a high-speed blender. The cellulose suspension was washed with water and stored at 4°C. The crystallinity of the cellulose substrates was estimated by x-ray powder diffraction (21) and showed that HCC is ϳ99% crystalline, whereas PASC is non-crystalline.
Cellulose Hydrolysis-The hydrolytic activities for HCC and PASC were determined by shaking each enzyme (final concentration of 1.4 M) and 0.1% substrate at 27°C in 50 mM sodium acetate (pH 5.0). Hydrolysis was evaluated at nine different time points up to 24 h, and the formation of soluble reducing sugars was determined with the p-hydroxybenzoic acid hydrazide method using cellobiose as a standard (22). The amount of released cello-oligosaccharides was also determined by highperformance anion-exchange chromatography at three or four time points basically as described previously (23). The standards were glucose (Fluka, Buchs, Switzerland); cellobiose, cellotetraose, and cellopentaose (Serva, Heidelberg, Germany); and cellotriose and cellohexaose (Associates Cape Cod, Inc.). In all cases, cellobiose was the major soluble product formed. Plots of the cellobiose produced were fitted to an exponential and linear equation, q(t) ϭ a⅐(1 Ϫ exp(Ϫb⅐t)) ϩ c⅐t, and a differential equation, v(t) ϭ a⅐b⅐exp(Ϫb⅐t) ϩ c, where a, b, and c are constants, t is the incubation time, q(t) is the product concentration, and v(t) is the production velocity.
Adsorption Analysis-Adsorption on HCC and PASC (0.1%, w/v) was measured under conditions similar to those used for hydrolysis with 1.4 M enzyme at pH 5.0 and 27°C. After 1 and 2 h of incubation, the mixtures were filtered through a Millex-GV 0.22-m membrane (Millipore) to terminate the reaction. Then, 700 l of 50 mM sodium acetate (pH 5.0) was added to 300 l of filtrate, and the protein concentration was measured using a Varian Cary Eclipse spectrofluorometer. A separate standard curve was prepared for each protein. The binding experiments were done in triplicate. The amount of adsorbed enzyme was calculated from the initial protein concentration, and the remaining cellulose contents at 1-and 2-h time points were calculated from the amount of cellobiose produced.
Molecular Dynamics Simulation-The initial coordinates of the TrCel7A catalytic domain were taken from Protein Data Bank structure 8CEL (2), and the structure of the W40A mutant was created by in silico mutation. The protonation states at pH 7.0 were determined using the PDB2PQR server (24), where the 10 disulfide bonds described in the Protein Data Bank were assigned to the relevant cysteine residues. To compare the behavior of the cellulose chain at the tunnel entrances of WT cat and W40A cat , the reducing end glucose (head) of cellononaose was initially positioned at subsite Ϫ7 (other parts were created automatically), which is the nonreducing end (tail) position in the original Protein Data Bank structure. The systems were then fully solvated with explicit solvent (including crystal water), and 19 Na ϩ counterions were added to obtain electrostatic neutrality. All simulations were performed using the AMBER 11 package (25). The AMBER ff03 force field was used for proteins. We employed the general AMBER force field with the AM1-BCC partial charges for the N-terminal pyroglutamic acid, the GLYCAM 06 force field for cellononaose, and the TIP3P model for water molecules. The systems were energetically minimized for 300 steepest descent steps and equilibrated for 1 ns with gradually reducing restraints. Finally, two 50-ns production runs were performed with different initial velocities for each system. The temperature and pressure were controlled using the Berendsen rescaling method (26), and the long-range electrostatic forces were calculated using the particle mesh Ewald method (27). The trajectory analysis was conducted using the AMBER module ptraj, and the snapshot structures were visualized with VMD (28). In addition, the molecular dynamics simulations starting form subsite Ϫ5 were performed using same methods as described above for both WT cat and W40A cat . seems to have a slightly increased substrate inhibition constant (K m2 ), which hints that the MULac substrate has also nonproductive binding modes involving subsite Ϫ7.

Hydrolysis of Soluble
Hydrolysis of Amorphous Cellulose-To study the hydrolysis of insoluble cellulose in detail, we first used amorphous cellulose (PASC) as a substrate and determined the amount of bound protein as well as the velocity of cellobiose production, which was calculated from the progress curves. As shown in Fig.  2A, all variants showed hydrolytic activity on PASC, and furthermore, the W40A mutant enzyme was Ͼ2-fold more efficient than the WT enzyme in terms of cellobiose production ( Table 2). The specific activities of the bound enzymes were also determined by dividing the cellobiose production rates by the amount of adsorbed enzyme at 1-and 2-h time points (Table 2). Because WT and W40A bound equally well to PASC, the specific activity of adsorbed W40A was 2.7 (1 h) and 2.2 (2 h) times greater than that of WT, indicating that Trp-40 slows down the hydrolysis of amorphous cellulose. Although the single-domain W40A cat version had quite low activity for PASC, its specific activity was similarly 2.7 (1 h) and 2.6 (2 h) times higher than that of WT cat , indicating that the negative effect of Trp-40 is independent of the presence or absence of the CBD. However, as shown in Table 2, W40A cat was significantly less well adsorbed on PASC compared with WT, W40A, and WT cat .
Hydrolysis of HCC-In contrast to PASC, cellobiose production by WT cat , W40A, and W40A cat was significantly decreased compared with WT when HCC was used as a substrate (Fig.  2B). The results indicate important roles for both Trp-40 and the CBD in the hydrolysis of crystalline cellulose. As shown in Table 2, the velocities of cellobiose production by W40A and WT cat on HCC were reduced to ϳ20 and 30% of that by WT, respectively. The specific activity of bound W40A was 45% (1 h) or 21% (2 h) of that of WT, whereas WT cat showed a specific activity that was 4.4 (1 h) or 2.6 (2 h) times higher than that of WT. The adsorption of W40A was 48% (1 h) or 73% (2 h) of that of WT. These results indicate that Trp-40 may play a role in initiating the degradation of HCC as well as in the processive hydrolysis of HCC, whereas the CBD hinders hydrolysis.
Molecular Dynamics Simulation-To examine the function of Trp-40 at the atomic level, molecular dynamics simulations were carried out for WT cat and W40A cat with cellononaose, with the reducing end glucose initially positioned at Trp-40 or Ala-40. Two 50 ns simulation runs were performed for each system (Fig. 3A), and the distances between the reducing end glucose and the catalytic carboxylic acid Glu-212 are plotted in Fig. 3B. In the case of WT cat , cellononaose entered the catalytic tunnel and finally penetrated ϳ11 Å within the tunnel, approximately two glucose units, toward the catalytic residue Glu-212 during the first simulation (supplemental Video S1). The cellononaose finally entered from subsite Ϫ7 to subsite Ϫ5. The

TrCel7A W40A Mutagenesis
second trial gave similar results, although the approach toward Glu-212 was much faster than in the first trial (supplemental Video S2). In contrast, in the case of W40A cat , the cellononaose did not move into the active site tunnel but diffused away from the alanine residue at subsite Ϫ7. The substrate chain bound on the back side of the catalytic domain in the first trial and on one of the loops forming the active site tunnel in the second trial (supplemental Videos S3 and S4). On the other hand, when the reducing end of the chain was initially positioned at subsite Ϫ5, both W40A cat and WT cat kept the cellononaose around the initial position, as shown in Fig. 3A. In the simulations of WT cat , glucose rings were stuck to subsites Ϫ5, Ϫ6, and Ϫ7 (supplemental Videos S5 and S6), which is similar to the simulations starting from subsite Ϫ7. However, the simulation of W40A cat starting from subsite Ϫ5 showed that the interactions at subsites Ϫ5 and Ϫ6 were strong enough to keep the cellononaose chain in the tunnel, which is very different form the simulations starting from subsite Ϫ7 (supplemental Videos S7 and S8). Interestingly, in the second trial of W40A cat , the cellononaose processed into the subsite tunnel much deeper than with WT cat (Fig. 3C). Consequently, these results indicate that Trp-40 at subsite Ϫ7 may assist entry of the cellulose into the subsite tunnel with a much shorter length of the free chain end, which initiates the processive reaction of TrCel7A.

DISCUSSION
The protein-carbohydrate interactions of carbohydrate-active enzymes are characterized by a network of hydrogen bonds and hydrophobic stacking interactions involving aromatic amino acid residues (29,30). Stacking interactions seem to be especially important in the case of enzymes and individual protein domains that act at the surface of insoluble substrates, e.g. carbohydrate-binding modules (31), chitinases (32), and cellulases (33). TrCel7A has a two-domain structure composed of a catalytic domain and a CBD, both of which have aromatic amino acid residues that serve to align the substrate-binding area and form hydrophobic stacking interactions with the glucosyl units of cellulose (2,31). In processive cellulose hydrolysis, the TrCel7A catalytic domain slides along the cellulose chain and releases the product (cellobiose) from subsites ϩ1 and ϩ2 and then proceeds to the next cellobiose unit from the reducing end. To investigate this process, we focused on the roles of TrCel7A Trp-40 and CBD in cellulose degradation. Trp-40 is located in an overhang at the entrance to the active site tunnel, and in the productive binding mode, it makes contact with the sugar ring (2).
With soluble MULac as a substrate, no significant difference in activity was detected, as expected ( Table 1). Elimination of the CBD did not significantly affect the PASC hydrolysis by the WT enzyme, whereas mutation of Trp-40 to Ala caused a clear increase in cellobiose production from amorphous substrates ( Table 2). Because PASC is an insoluble substrate, the adsorption behavior was also compared and is summarized in Scheme 1. The results indicate that the CBD had a very small effect, if any, on the adsorption of the WT enzyme on PASC, which was also an expected result. However, W40A cat bound less strongly to PASC compared with WT cat and the CBD-containing mutant (W40A). This suggests that adsorption of the TrCel7A catalytic domain on amorphous cellulose is dependent on Trp-40. The CBD can, however, compensate for the binding defect caused by the W40A mutation. Concerning the specific activity of the adsorbed enzymes on PASC, Trp-40 had a negative effect, as shown in Scheme 1. We assume that this is due to the stacking of a substrate at subsite Ϫ7, which interferes with the cellulose chain slide within the tunnel and with the dissociation of the catalytic domain from a cellulose chain.
In contrast, the hydrolysis of HCC showed completely different behavior compared with MULac and PASC. Enzyme adsorption was clearly promoted by the CBD, whereas Trp-40 had less of an effect on the overall binding. However, the specific activity of the adsorbed enzyme on HCC was greatly enhanced by the presence of Trp-40 but hindered by the CBD. This is an important finding because there is likely to be a very sensitive balance between Trp-40 and the CBD affecting the processes of processivity and adsorption. Considering the molecular dynamics simulation results, Trp-40 should have a particularly important role when a limited number of reducing end glucose units are available to the active site. The essential difference between amorphous and crystalline celluloses is an availability of the free chain ends. Therefore, HCC has only a  small number of glucose units free at the chain end, whereas PASC has longer chain ends available. The free reducing end of the chain might not reach a deep position of the subsite without Trp-40, which is critical for HCC degradation. In contrast, the chain ends of PASC easily reach subsite Ϫ5, and Trp-40 is unnecessary for chain loading. These results are also supported by a previous molecular dynamics study that calculated the binding energy of each subsite (3,34). Concerning the role of the CBD, the population of the adsorbed enzyme is clearly larger in the presence of the CBD, as can be seen when comparing the adsorption of WT cat and WT. In contrast, the CBD lowers the specific activity of the adsorbed enzyme. There may be two reasons for this. 1) The CBD may restrict the intramolecular processive movement of the enzyme on cellulose surface.
2) The CBD may lead to the nonproductive binding of enzyme on places where the catalytic module does not find a free chain end. In our high-speed atomic force microscopy study, we observed the apparent movement of WT cat on HCC and suggested that the major contribution of the CBD is simply to increase the concentration of adsorbed enzyme on the crystalline substrate (10). The present experiments with WT and WT cat further suggest that CBD-mediated adsorption involves nonproductive binding and/or disturbance of the hydrolytic activity, which is seen as reduced specific activity of the adsorbed enzyme. In our previous high-speed atomic force microscopy work, we observed a "wriggling" movement of the W40A mutant enzyme on the HCC surface and speculated that this is due to an inability to recruit the substrate chain into the active site tunnel (10). Our molecular dynamics simulation results presented here also support this hypothesis, and the biochemical data suggest a function of Trp-40 in initiating the processive sliding movement on HCC. The importance of this Trp residue in crystalline cellulose degradation is further supported by the fact that this residue is conserved among the sequences of GH7 family cellobiohydrolases (CAZy Database) as shown in supplemental Fig. S2 (35). Furthermore, there is a Trp residue at the subsite entrance that is conserved in GH6 family cellobiohydrolases. It has been shown previously that T. reesei GH6 family Cel6A cellobiohydrolase variants with this corresponding Trp residue mutated to alanine or aspartic acid have clearly decreased hydrolysis activity on bacterial microcrystalline cellulose but not PASC (36). This suggests that a tryptophan residue located at the entrance of the subsite is essential for substrate loading in all cellobiohydrolases that degrade crystalline celluloses.
In conclusion, this results of this study indicate that the CBD of TrCel7A serves to increase the enzyme concentration on the HCC surface, but at the expense of reducing the specific activity of the adsorbed enzyme. The main function of the conserved Trp-40 residue at the edge of the subsite tunnel appears to be recruitment of cellulose chain reducing ends into the active site tunnel and initiation of processive hydrolysis of the cellulose chain on HCC. This is important because long free cellulose chain ends are not frequently available on HCC, but again, there is a tradeoff due to the stacking interaction of Trp-40, which impedes progression of the cellulose chain within the active site tunnel. Consequently, removal of Trp-40 leads to more efficient degradation of less crystalline substrates.