mutational effects on the catalytic mechanism of cellobiohydrolase i from trichoderma...

Published: April 08, 2011

r 2011 American Chemical Society 4982 dx.doi.org/10.1021/jp200384m | J. Phys. Chem. B 2011, 115, 4982–4989

ARTICLE

pubs.acs.org/JPCB

Mutational Effects on the Catalytic Mechanism of CellobiohydrolaseI from Trichoderma reeseiShihai Yan, Tong Li, and Lishan Yao*

Key Lab of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, PR China

1. INTRODUCTION

Cellulose, the most abundant renewable biomass source onearth, is an insoluble homopolymer linked by unbranched β-1,4glycosidic bonds. The biodegradation of cellulose gives birth toglucose, which has crucial functions in biological systems.Furthermore, glucose can be fermented to ethanol or otherchemicals to replace nonrenewable petroleum. The growingneeds for utilization and recovery of natural resources haveprompted extensive scientific efforts toward a better recognitionof the basic mechanisms behind cellulose degradation. Cellu-lases, the enzymes biodegrading the cellulose, usually consist oftwo domains, a large catalytic domain (CD) and a smallcarbohydrate-binding module (CBM), combined by a glycosy-lated linker peptide.1,2 Efficient enzymatic conversion of crystal-line cellulose is important for an environmentally sustainablebioeconomy.3 The major bottleneck in the conversion of crystal-line cellulose-based biomass to ethanol or other chemicals is thelow activity of cellulases. It is now widely accepted that a betterunderstanding of the cellulase-catalyzed hydrolysis of cellulose ishighly desired and remains critical to enabling a successfulbioethanol industry. The catalytic mechanisms for glycosylhydrolases (GHs) are described in detail in several excellentreviews.2,4�12

Cellulolytic enzymes, like all GHs, hydrolyze glycosidic bondsvia the mechanism of general acid/base catalysis, whereby twocritical amino acid residues are needed, a proton donor and anucleophilic assistant. Generally, the two residues playing theseroles are glutamic and aspartic acid. So far, two distinct reaction

mechanisms for the GH superfamily enzymes have been pro-posed: inverting and retaining hydrolysis (Scheme 1). Thisscheme demonstrates that several covalent and noncovalentinteractions are involved in the bond breaking and formationprocesses during the enzyme catalysis with both mechanisms. Inthe first mechanism (inverting hydrolysis), the RO group gets aproton from the protonated acidic residue and shifts away fromthe cellulose chain. Simultaneously, the negatively charged acidicresidue activates water for nucleophilic attack on the carbocationcenter by electrostatic attraction.10,13�16 It has been suggested,based on the experimental observations and computationalresults, that the second mechanism is composed of two separatesteps.13,14,17,18 Initially, along with proton transfer from thedonor residue to the leaving RO group, a covalent glycosyl-enzyme intermediate is generated by the nucleophilic attack ofthe negatively charged residue on the glycosyl carbocationcenter. The original proton donor activates a water moleculeby capturing one of its protons in the subsequent step. Thehydrolysis product is released from the active site by nucleophilicattack of the water hydroxyl group. Accordingly, the retainingmechanism includes the separate glycosyl decomposition andhydrolysis steps. The predominant structural difference betweeninverting and retaining glycosidases is the distance between theproton donor and the nucleophile. The average distance between

Received: January 13, 2011Revised: March 30, 2011

ABSTRACT: QM/MD simulations are performed to study mutationaleffects on the glycosylation step of the oligosaccharide hydrolysis catalyzedby Trichoderma reesei cellobiohydrolase I. The potential of mean forcealong the reaction pathway is determined by the umbrella samplingmethod. A detailed mechanism is developed to illustrate the decrease inactivity of the mutants. Our calculations demonstrate that (1) the E212Qmutation increases the overall activation barrier by ∼4.0 kcal/mol, whilethe D214Nmutation causes∼0.4 kcal/mol increase of the barrier, and (2)there is only one transition state identified in the wild type (WT) andD214N mutant, while two transition states exist in the E212Q mutant forthe glycosylation process. The results explain the experimental observationthat the E212Q mutant loses most of its hydrolysis capability, while theD214N mutant only reduces it slightly compared to the WT. Furtheranalysis suggests that the proton transfer from Glu217 to O4 and theglycosidic bond cleavage between subsitesþ1 and�1 are concerted, facilitating the subsequent nucleophilic attack of Glu212 on C1

0in subsite�1. Our QM/MD study illustrates the importance of the prearrangement of the active site and provides atomic details ofthe enzymatic catalytic mechanism.

4983 dx.doi.org/10.1021/jp200384m |J. Phys. Chem. B 2011, 115, 4982–4989

The Journal of Physical Chemistry B ARTICLE

the catalytic residues is 5( 0.5 Å for the retaining and 10( 0.5 Åfor the inverting enzymes.10,11,19 The large separation is neces-sary in inverting enzymes to allow the substrate and water to bindsimultaneously between the carboxyl groups.

Cel7A from Trichoderma reesei (T. reesei), also known ascellobiohydrolase I (CBHI), hydrolyzes the β-1,4 linkages of acellulose chain from its reducing end via a retaining mechanismliberating the product, which consists of roughly 63% β-cellobioseand 37%R-cellobiose due to the anomeric equilibrium.1,20�26 It hasbeen confirmed that Cel7A provides most hydrolytic power amongthe T. reesei enzymes contributing to cellulose degradation.27 Thestructural determination of the CD has paved the way for detailedinvestigations of the catalytic mechanism.17,28,29 Taking the positionof the cleaved glycosidic bond as the reference point, the glucosyl-binding subsites are numbered according to the generally acceptedsubsite-naming convention.30 The active site of CBHI containsthree carboxylate residues (Glu212, Asp214, and Glu217) that appearstrategically positioned for participation in catalysis (Figure 1). Theproximity of Glu217 to the O4 atom in subsiteþ1 suggests that thisresidue may act as a general acid by donating a proton, while Glu212

plays the role of a nucleophile during a double displacement at theanomeric carbon atom C1

0. The third residue, Asp214, between thetwo glutamic acid residues, may be responsible for the correctpositioning and protonation of Glu212. To assess the importance ofthese three carboxylate residues, several studies have been carriedout by means of site-directed mutagenesis, where each carboxylateresidue was replaced by its isosteric amide.28,29 The most dramaticeffects were observed for the E217Q and E212Q mutations, whilethe smallest effect was shown for the D214N mutant, which stilldisplays some activity. Although the structures have been deter-mined in atomic detail, the intrinsic mechanism for the distinctactivity reduction of the mutants remains unknown. To understandthe mutational effect on enzyme catalysis, in principle one has to

study the whole process, including substrate binding, the catalyticreaction (glycosylation and deglycosylation), and product release.However, if the step controlling the reaction rate is known(assuming it is the same for the wild type and mutants), one cangreatly reduce the amount of work by studying only this step. It hasbeen proposed that glycosylation is rate-limiting for the substrate2-chloro-4-nitrophenyl-β-lactoside.28 Therefore, illustrating themech-anism of the glycosylation step is critical to understand themutational effects.Here, we apply a quantummechanicsmoleculardynamics (QM/MD) simulation method to study this process forthe wild type and the E212Q andD214Nmutants. E217Qwas notstudied here since it has a similar effect as the E212Q mutant. Tobetter mimic the hydrolysis of cellulose, we use a cellulose nanomeras the substrate.

Computational modeling approaches can provide a powerfultool for understanding enzyme catalysis.17,31 QM/MD methodsthat combine the accuracy of quantum mechanics (QM) and theefficiency of molecular mechanics (MM) are an effective tool tounderstand characteristics of macromolecular systems.32 It allowsfor chemical bond breaking/formation and dynamics of the activecenter while including the effects of the fluctuating protein environ-ment. In this work, QM/MD simulations for the above-describedenzyme�substrate complexes were performed. The umbrella sam-pling method was used to move along a reaction coordinateappropriate to simulate the glycosylation step, and the weightedhistogram analysis method (WHAM)33�35 was utilized to calculatethe potential of mean force profile along this reaction coordinate.

The simulations were carried out on a modeled structure,8CEL, which is based on the protein X-ray crystal structures of theE217Q mutant complexed with cellohexaose and the E212Qmutant complexed with cellotetraose and cellopentaose, respec-tively. Two mutants, E212Q and D214N, were generated asdescribed in Section 2. The computational details, including thepreparation of theWT andmutant QM/MD structural models andthe description of the hybrid QM/MDmethodology, are presentedin Section 2. Section 3 collects the results and discusses themechanistic details. Our conclusions are summarized in Section 4.

2. COMPUTATIONAL DETAILS

Preparation of the Simulation System. The initial coordi-nates of the WT were prepared according to a theoretical model,PDB entry 8CEL (Figure 1).29 The E212Q and D214N mutantswere generated computationally, Glu212 Oε2 f Gln212 Nε2 forE212Q and Asp214 Oδ2 f Asn214 Nδ2 for D214N, respectively.

Scheme 1

Figure 1. Schematic representation of T. reesei Cel7A (PDB entry8CEL) with a cellooligomer bound. The acidic amino acid residue,Glu217, is above the cellulose chain, acting as the proton donor. Glu212,the nucleophile, is below the active site. Asp214 is behind the chain.



E217 and D214 were assumed to be neutral based on the X-raystructure.28,29 All prepared systems were neutralized by addingNaþ ions with the Amber tool and were solvated in a rectangularbox with a 12 Å buffer distance between the solvent box wall andthe nearest solute atoms. For water molecules, the TIP3P modelis utilized. Each of these three systems consists of∼65 000 atoms.Throughout this work, all of the simulations were performedwith the AMBER11 molecular dynamics package.36 The systemwas first minimized and equilibrated and then simulated for 1 nswith periodic boundary conditions, constant temperature at 300K,and pressure of 1 atm. The FF99SB Amber37 and GLYCAM_06force fields38 are employed to model the enzyme and the sub-strate, respectively.Hybrid QM/MD Approach. The active site of CBHI contains

three carboxylate residues, Glu212, Asp214, and Glu217. Thecellulose hydrolysis at the active site is initialized by the protontransfer from the donor residue Glu217 carboxyl group to O4,which holds the glucosyl residue to the reducing side of theglycosidic bond. The system was partitioned into QM and MMregions. The QM region, including three carboxylate residues aswell as the glycosyl rings in subsites �1 and þ1, was treated bythe PM3 semiempirical method.39,40 TheMM region contains allother protein residues, solvent water molecules, as well as theglycosyl rings except the subsites �1 and þ1. The last snapshotfrom the classical MD simulation was employed for the subse-quent QM/MD simulations (with the step size of 0.001 ps). Theumbrella sampling and weighted histogram analysis method(WHAM)33�35 were utilized to determine the free-energyprofile for the proton transfer and the nucleophilic attack. TheGlu217 Hε2 proton transfer to the O4 atom was divided into∼30windows (with the Hε2�O4 distance as the reaction coordinate),each of which was simulated for 50 ps. Totally,∼1.5 ns QM/MDsimulations were carried out to characterize the proton transferprocess in the WT and mutants, E212Q and D214N. Thenucleophilic attacking process, corresponding to the bond for-mation between C1

0 in subsite�1 and Oε1 of Glu212 (or Gln212),was simulated with about 35 windows (with the C1

0�Oε1

distance as the reaction coordinate) and 50 ps for each window.Harmonic restraints with force constants of 400 kcal/mol Å2

were employed for proton transfer reaction and the subsequentnucleophilic attacking process by shortening the Hε2�O4 andC1

0�Oε distances, respectively. The configurations after 10 ps ineach window were collected for data analysis. The probabilitydistribution along the reaction coordinate was determined foreach window and pieced together with the WHAMapproach,15,16,41�44 to calculate the potential of mean force(PMF) profile. The error analysis was performed by dividingthe gathered data into two equal blocks.

3. RESULTS AND DISCUSSION

QM/MD simulations were carried out after classical molecu-lar dynamics equilibration, which was performed for the fullysolvated WT and the mutants at the NPT condition (300 K and1 atm). The primary geometric parameters of the enzyme substratecomplex (ES), the transition state of proton transfer process(TSP), the intermediate after proton transfer (IM), the transitionstate of nucleophilic attacking reaction (TSN), and the glycosyl-enzyme intermediate (GI) are all collected in Tables 1�3, asextracted from the QM/MD simulations for the WT andmutants. The PMFs for the WT and the mutants determinedby the QM/MD simulations and umbrella sampling method are

shown in Figure 2. The variation of the distance of O4 and C10,

O4�C10, in the WT and mutants during the glycosylation

process is presented in Figure 3. Figure 4 illustrates the enzyme�substrate interactions in the tunnel of CBHI.3.1. Potential of Mean Force. To reveal the mechanism

behind the mutants’ activity loss, the PMFs of the glycosylationprocess, which is composed of the proton transfer and thenucleophilic attack, for the WT and the mutants are determinedemploying theQM/MD simulations and umbrella sampling. Theobtained PMFs are shown in Figure 2, which was drawn bysetting the free energies of substrate complexes at zero. It shouldbe emphasized that this origin is used only to compare the PMFbarriers more easily. The binding free energies of the threesubstrate complexes are not determined here and most likely aredifferent. As a result, the direct comparison of the absolute PMFof the three complexes is not meaningful. For theWT, a high freeenergy activation barrier of 32.6 kcal/mol needs to be overcomeduring the proton transfer process. This barrier is higher than

Table 1. Primary Geometric Parameters (Distances in Å) ofthe Enzyme Substrate Complex (ES), the Transition State ofthe Proton Transfer Process (TSP), the Intermediate afterProton Transfer (IM), the Transition State of the Glycosyla-tion Reaction (TSN), and the Enzyme Glycosyl Intermediate(GI) for the WTa

ES TSP IM TSN GI

Nη1(Arg107)�O3(�2) 2.90 2.89 2.89 2.90 2.90

Nη2(Arg107)�O6(�3) 3.05 3.02 3.02 4.72 4.72

Oη(Tyr145)�O2(�2) 2.89 2.85 2.85 2.79 2.79

Oδ2(Asp179)�O6(�3) 2.55 2.56 2.56 2.54 2.54

Oη(Tyr247)�O6(�2) 3.87 4.18 4.18 3.91 3.91

O(Asp368)�O2(�3) 3.19 3.23 3.23 3.10 3.10

Oε2�Hε2(Glu217) 0.96 1.04 2.08 2.14 1.98

Hε2(Glu217)-O4 2.28 1.24 0.97 0.97 0.97

Oε2(Glu217)�O60 3.97 3.62 2.98 2.83 2.94

O4�C10 1.42 1.47 3.41 3.26 3.89

C10�Oε1(Glu212) 5.02 4.96 3.51 3.68 2.82

C10�Oε2(Glu212) 3.60 3.48 2.15 2.34 1.44

Oε2(Glu212)�O20 2.74 2.74 3.06 3.02 3.22

Oε1(Glu212)�O20 3.16 3.18 2.70 2.70 2.64

Oδ2(Asp214)�Oε2(Glu212) 2.68 2.68 2.69 2.69 3.43

Nε2(His228)�O3 2.86 3.27 3.61 3.39 3.38

Nε2(His228)�Oδ1(Asp214) 3.09 3.17 3.03 3.06 3.08

Oδ1(Asp214)�O3 3.24 2.66 3.16 3.12 3.18

N(Ser174)�Oε1(Glu212) 2.90 2.89 3.07 3.08 3.30

Oγ(Ser174)-Nδ1(His228) 3.50 3.57 2.96 3.24 2.88

Oγ(Ser174)�Nε2(Gln175) 3.06 2.99 3.03 3.11 3.21

Nε2(Gln175)�O20 3.23 3.50 3.22 3.21 3.14

Oε1(Gln175)�Nη1(Arg251) 3.11 3.28 3.07 3.10 3.10

Oε1(Gln175)�Nη2(Arg251) 2.88 2.89 2.88 2.87 2.85

Oδ1(Asp259)�Nε(Arg251) 2.84 2.85 2.84 2.86 2.84

Nη1(Arg394)�O1(þ2) 3.37 3.40 3.73 3.89 3.67

Nη1(Arg394)�O5(þ2) 2.95 2.90 2.93 3.24 2.95

Nη1(Arg394)�O6(þ2) 4.21 4.05 4.05 4.23 3.94

Nη2(Arg394)�O1(þ2) 3.10 2.95 3.06 3.12 3.01

Nη2(Arg394)�O5(þ2) 3.96 3.84 3.65 3.52 3.64

Nη2(Arg394)�O6(þ2) 5.41 5.34 5.19 4.89 5.00aThe data are averaged over the snapshots.



that obtained by Li et al.18 for theWTCBHI-catalyzed glycosyla-tion of cellobiose. In Li et al.’s work, the energy barrier of 14.1kcal/mol is obtained with a simplified model system includingthree residues E212, D214, and E217 and cellobiose employingthe density functional theory (DFT) method (B3LYP/6-31G(d,p)45). Several factors can contribute to this discrepancy.The three residues in the DFT calculations were allowed tomovefreely in the search for the transition state, which is artificial, sincethe position of these residues and cellobiose is restrained bythe enzyme. In fact, the barrier increases to 23.9 kcal/mol, when thewhole protein is included, closer to that obtained in this work.Furthermore, the barrier from Li et al.’s work is in energy while inour work it is free energy, and different QM methods used mayalso contribute to the barrier difference.Compared to the barrier of proton transfer, the barrier of the

nucleophilic attacking step is small, only ∼0.4 kcal/mol. Thus,the whole process from the ES to GI can be taken as a singletransition state (TSP) reaction, and the proton transfer process is

rate-limiting for theWT. The error analysis indicates that the freeenergy profile converges well, suggesting the sampling issufficient.Similar to the WT, the glycosylation of D214N essentially has

one transition state (TSP) as seen in Figure 2. The free energyactivation barriers of the proton transfer process and thenucleophilic attack reaction are higher for the D214N mutantby ∼0.4 kcal/mol than those of WT. The slightly higher energybarrier indicates a somewhat lower catalytic activity. In addition,the GI of D214N is lower in energy by about 4.0 kcal/molthan its ES, as is favorable for the glycosylation step. The small0.4 kcal/mol barrier increase corresponds to the kcat loss of 50%based on the Arrhenius equation, close to the experimental datawhere after 100 h incubation of bacterial microcrystalline cellu-lose (BMCC) with the D214N mutant the amount of reducingsugar is ∼35% less than that from incubation with the WT. It isworth noting that the comparison is very qualitative since theproduct quantity difference in experiment also depends on the

Table 2. Primary Geometry Parameters (Distances in Å) ofthe Enzyme Substrate Complex (ES), the Transition State ofthe Proton Transfer Process (TSP), the Intermediate afterProton Transfer (IM), the Transition State of the Glycosyla-tion Reaction (TsN), and the Enzyme Glycosyl Intermediate(GI) for the E212Q Mutanta

ES TSP IM TSN GI

Nη1(Arg107)�O3(�2) 2.93 2.90 2.98 2.89 2.87

Nη2(Arg107)�O6(�3) 3.23 3.04 3.17 3.14 3.09

Oη(Tyr145)�O2(�2) 2.79 2.82 2.81 2.84 2.84

Oδ2(Asp179)�O6(�3) 2.64 2.68 2.62 2.62 2.61

Oη(Tyr247)�O6(�2) 390 4.12 4.52 4.38 4.58

O(Asp368)�O2(�3) 2.92 2.75 2.82 2.71 2.69

Oε2�Hε2(Glu217) 0.98 1.78 1.85 3.38 3.10

Hε2(Glu217)�O4 1.82 1.02 0.96 0.95 0.95

Oε2(Glu217)�O60 4.63 3.11 3.06 4.56 4.58

O4�C10 1.44 4.05 3.08 5.13 5.11

C10�Oε1(Gln212) 3.46 3.33 2.80 2.09 1.47

C10�Nε2(Gln212) 4.80 3.67 4.22 3.34 2.99

Nε2(Gln212)�O20 3.86 3.70 4.04 3.04 2.81

Oε1(Gln212)�O20 3.52 4.33 3.55 3.29 3.08

Oδ2(Asp214)�Oε1(Gln212) 2.69 2.69 2.75 2.66 2.78

Nε2(His228)�O3 3.17 3.29 3.39 3.32 3.32

Nε2(His228)�Oδ1(Asp214) 3.04 3.14 2.99 2.97 2.95

Oδ1(Asp214)�O3 3.08 3.18 3.33 3.34 3.30

N(Ser174)�Oε1(Gln212) 5.88 5.68 5.75 5.93 5.99

Oγ(Ser174)�Nδ1(His228) 2.89 3.05 2.99 3.01 3.15

Oγ(Ser174)�Nε2(Gln175) 3.45 3.72 3.26 3.58 5.20

Nε2(Gln175)�O20 3.16 3.40 3.33 3.54 3.66

Oε1(Gln175)�Nη1(Arg251) 4.85 4.57 4.73 4.79 4.89

Oε1(Gln175)�Nη2(Arg251) 4.70 4.60 4.60 4.66 5.14

Oδ1(Asp259)�Nε(Arg251) 3.21 3.19 3.10 4.44 4.51

Nη1(Arg394)�O1(þ2) 3.88 3.99 3.91 3.85 3.95

Nη1(Arg394)�O5(þ2) 3.05 3.17 3.06 3.05 3.10

Nη1(Arg394)�O6(þ2) 3.10 2.97 3.01 2.94 2.93

Nη2(Arg394)�O1(þ2) 2.96 2.96 2.94 2.91 2.98

Nη2(Arg394)�O5(þ2) 3.36 3.30 3.28 3.26 3.19


Table 3. Primary Geometry Parameters (Distances in Å) ofthe Enzyme Substrate Complex (ES), the Transition State ofthe Proton Transfer Process (TSP), the Intermediate afterProton Transfer (IM), the Transition State of the Glycosyla-tion Reaction (TSN), and the Enzyme Glycosyl Intermediate(GI) for the D214N Mutanta

reactant TSP IM TSN GI

Nη1(Arg107)�O3(�2) 2.88 2.95 2.92 2.90 2.90

Nη2(Arg107)�O6(�3) 2.95 3.28 3.03 3.14 3.14

Oη(Tyr145)�O2(�2) 2.85 2.86 2.85 2.98 2.93

Oδ2(Asp179)�O6(�3) 3.51 2.59 2.66 2.54 2.55

Oη(Tyr247)�O6(�2) 3.28 2.77 2.75 2.97 2.75

O(Asp368)�O2(�3) 3.85 3.54 3.59 3.56 3.66

Oε2�Hε2(Glu217) 0.96 1.66 1.79 1.78 1.81

Hε2(Glu217)�O4 1.97 1.04 0.98 0.97 0.96

Oε2(Glu217)�O60 3.83 3.15 3.27 3.46 3.05

O4�C10 1.43 2.88 3.47 4.92 4.91

C10�Oε1(Glu212) 4.34 3.51 3.39 2.29 1.39

C10�Oε2(Glu212) 3.42 2.92 2.76 4.26 3.36

Oε2(Glu212)�O20 3.19 3.47 3.43 4.50 4.17

Oε1(Glu212)�O20 2.84 2.76 2.71 3.34 3.07

Nδ2(Asn214)�Oε2(Glu212) 4.82 4.92 4.80 4.75 4.70

Nε2(His228)�O3 3.16 3.23 3.21 3.18 3.08

Nε2(His228)�Oδ1(Asn214) 5.59 5.45 5.21 4.73 5.15

Oδ1(Asn214)�O3 3.23 3.23 2.93 2.82 3.24

N(Ser174)�Oε1(Glu212) 3.12 3.08 3.14 6.17 6.07

N(Ser174)�Nδ1(His228) 3.61 3.57 3.58 2.91 3.00

N(Ser174)�Nε2(Gln175) 2.95 2.99 2.98 3.24 3.42

Nε2(Gln175)�O20 3.29 3.47 3.66 3.62 3.60

Oε1(Gln175)�Nη1(Arg251) 3.24 3.41 3.59 3.62 3.49

Oε1(Gln175)�Nη2(Arg251) 2.89 2.92 2.91 3.07 2.94

Oδ1(Asp259)�Nε(Arg251) 3.42 3.64 3.48 3.60 3.59

Nη1(Arg394)�O1(þ2) 4.01 4.33 4.37 4.13 4.22

Nη1(Arg394)�O5(þ2) 2.96 3.13 3.15 3.00 3.06

Nη1(Arg394)�O6(þ2) 3.18 2.95 2.93 2.97 2.92

Nη2(Arg394)�O1(þ2) 3.12 3.19 3.11 3.10 3.11

Nη2(Arg394)�O5(þ2) 3.49 3.37 3.27 3.41 3.37




binding kinetics and thermodynamics of the substrate/productto the WT and D214N enzyme, which are unknown.For themutant E212Q, the free energy activation barrier of the

proton transfer process is 31.2 kcal/mol, slightly lower than thatof the WT. However, there is a higher barrier (8.4 kcal/mol) inthe nucleophilic attacking step. The energy of the nucleophilicattacking transition state (TSN) is higher bymore than 4.0 kcal/molthan the proton transfer transition state, TSP. Therefore, twotransition states (TSP and TSN) exist during the process exploredhere, and the TSN determines the reaction rate. Another pointshould be mentioned that the GI of the E212Q mutant isthermodynamically unfavorable because its energy is 13 kcal/molhigher than its ES. Therefore, the hydrolysis rate should be slowerthan that catalyzed by the WT. The experimental study showed thatafter 100 h incubation of BMCCwith the E212Qmutant the amountof reducing sugar is only ∼6% of that yielded from the incubationwith the wild type enzyme,28 which corresponds to a barrier increaseof 1.7 kcal/mol if the mutation only impacts kcat. This value iscomparable to the barrier increase obtained in our work.The above analyses illustrate qualitatively the loss of the

enzyme activity in the mutants, which confirms the previousexperimental results.28,29 The small variations in PMF during theproton transfer process illustrate the slight influence brought bymutations, while distinct effects can be found for the nucleophilicattacking reaction and the GI free energy relative to the ES. Fromthe structural point of view, it is reasonable since residues 212 and214 are close to the nucleophilic attack reaction center and aresomewhat removed from the proton transfer site. The second highfree energy barrier of the E212Q mutant is conceivably caused bythe net charge change of this residue from�1 to 0. Since the targetof the nucleophilic attack C1

0 is positively charged, the weakening ofthe electrostatics between Oε (Q212) and C1

0 can no longerovercome the repulsion between the two atoms and correspondingfragments as they approach each other. Therefore, a new energybarrier is observed for E212Q but not for the WT and D214N.Amore detailed geometric analysis is provided below to illustrate themutational effects on the enzyme activity.3.2. Active Site Geometry. Various proteins carry out differ-

ent biological functions owing to their special geometric arrange-ments. The biological function of a protein is an inherentproperty of the structure, which is the basis of and tightly relatedto the function of the molecule. For the hydrolysis of cellulose byCel7A, the reaction mechanism is determined by the coupling

mode and the conformation of the enzyme�substrate complexwith the active site located between subsites�7 andþ2. The twocritical residues, a proton donor and a nucleophilic assistant, liebetween subsites �1 and þ1. Therefore, the interactionsapproximate to these two subsites are vital and play key rolesduring the hydrolysis process. Here, the couplings in subsites�3, �2, þ2, as well as �1 and þ1 are considered in detail.3.2.1. Binding Subsites �3 and �2. The hydrogen bond

interactions are summarized in Table 1 for the WT. The Oδ2-(Asp179)�HO6(�3) hydrogen bond, betweenOδ of the Asp179side chain carboxyl group and the O6H hydroxyl of glucosyl insubsite�3, which was observed experimentally by Tones et al.,29

is a short, strong hydrogen bond (SSHB) in the ES state. TheSSHB characteristic of this interaction remains during the wholeproton transfer and the nucleophilic attack processes, as reflectedby the short distance in Table 1. O6 in subsite�3 participates inanother hydrogen bond with Nη2 of the Arg107 residue, Nη2H-(Arg107)�O6(�3), which weakens in the GI state. The thirdhydrogen bond, found in subsite �3 between the O2 hydroxylgroup and backbone carbonyl O of residue Asp368, O-(Asp368)�HO2(�3), changes slightly during the reaction. Insubsite�2, O3 of glucosyl forms a hydrogen bond with the Nη1Hof Arg107. This hydrogen bond can be observed from the ES tothe GI state. During the proton transfer and nucleophilic attackprocesses, two tyrosine residues, Tyr145 and Tyr247, located onthe opposite sites of subsite�2, take part in the hydrogen bondswith O2 and O6, respectively, with the former stronger than thelatter. Besides these two hydrogen bonds, glycosyl in subsites�2also aligns approximately parallel to the tryptophan ring ofTrp367. Such an arrangement minimizes the interaction energyand should be responsible for the twist of the glycosyl in thissubsite to be almost perpendicular to that in subsite �4. All thehydrogen bonds and van der Waals interactions discussed herecan be found in the E212Q and D214Nmutants (Tables 2 and 3).It can be inferred that the influence of mutations to subsites �3and �2 as well as their ambient interactions is small.3.2.2. Binding Subsites �1 and þ1. One of the most

prominent phenomena that occurs between subsites �1 andþ1 is the glycosidic bond O4�C1

0 cleavage as a result of theprotonation of O4 by H

ε2(Glu217). The variations of the glyco-sidic bond, O4�C1

0, in the WT and mutants along the protontransfer coordinate and the nucleophilic attack reaction arepresented in Figure 3. Plots (a)�(c) illustrate the variations

Figure 2. Complete free energy profile for the proton transfer process (a) and nucleophilic attacking reaction (b) determined by the QM/MDsimulations and umbrella sampling. The statistical error is estimated by averaging the free energy difference between 10 and 30 ps and 30�50 ps. Thedifferences of the GI PMF values in the three enzyme complexes reflect different relative product binding affinity from that of the reactant.



during the proton transfer process, and the changes in thenucleophilic attack step are collected in (d)�(f). When thelength of Hε2(Glu217)�O4 approaches 1.0 Å, the O4�C1

0 bondlengthens significantly. In other words, the covalent O4�C1

0bond cleavage is concerted with the Hε2(Glu217)�O4 bondformation, after which the cellobiose dissociates from the cellu-lose. Accompanying the O4�C1

0 bond cleavage, the Cδ-(Glu212) 3 3 3C1

0 distance decreases distinctly, favoring thesubsequent nucleophilic attack. This phenomenon is observedin the WT and mutants. Therefore, the mutations maintain theproperty that the proton transfer and the glycosidic bondO4�C1

0 cleavage occur synchronously, followed by the nucleo-philic attack. This is consistent with the earlier QM/MMresults.17,18,32,46 In WT, the O4�C1

0 distance lengthens alongwith the decrease of the Cδ(Glu212)�C1

0 distance during the

nucleophilic attack of Glu212, which indicates that the cellobiosemoves away from the active site. A similar increase of the distanceis observed in E212Q, while in the D214N mutant it fluctuatesaround 4.8 Å during the nucleophilic attack process. At the end ofthe glycosylation reaction, a novel covalent bond is generatedbetween C1

0 and Oε2 of the Glu212 carboxyl group (Tables 1, 2,and 3). The tables also indicate that this carboxyl group interactswith the O2H group in subsite�1 during the nucleophilic attack.In the WT, when the distance between Hε2 (Glu217) and O4

decreases from 2.28 to 0.97 Å, the Oε2�Hε2 distance of residueGlu217 increases continuously. This proton covalently links to O4

in the IM state (Table 1). The variation of the newly formedHε2(Glu217)�O4 bond is very small in the subsequent nucleo-philic attack process. The same phenomenon can also beobserved in the E212Q and D214N mutants. The electrostatic

Figure 3. Variations of theO4�C10 distance with the simulation time. (a)�(c) represent the distance changes during the proton transfer process for the

wild-type enzyme�substrate complex and themutants, respectively. (d)�(f) denotes the alterations during the nucleophilic attack process, respectively.



interaction between Oε2(Glu217) and O60H in the ES state

changes into the hydrogen bond in the IM state. This hydrogenbond still can be found after glycosylation, as is supported by theexperimental X-ray structures.29 The same phenomenon occursin D214N, while the hydrogen bond generated in IM disappearsin the GI state of the E212Q mutant (Tables 1, 2, and 3). Thesevariations are coincidental with the changes of the PMFs, butwhether this interaction is related to the free energy barrierremains unclear. Another point should be mentioned that, inagreement with the previous report,28 a strong hydrogen bondexists between Asp214 (protonated) and Glu212 carboxyl groupsin the ES of WT. This hydrogen bond is maintained until theformation of the covalent C1

0�Oε2(Glu212) bond in the GI statewhere it is weakened significantly (Table 1). This observationsuggests that Oδ2 of Asp214 positions Oε2 of Glu212 to facilitatethe nucleophilic attack process. The Oδ2H(Asp214)�Oε1-(Gln212) hydrogen bond is maintained throughout the glycosyla-tion step in the E212Q mutant. However, its counterpart in theD214N mutant, the Oδ2H(Asn214)�Oε1(Glu212) hydrogenbond, cannot be observed.Besides the hydrogen bonds mentioned above, a complex

hydrogen bond network is also observed in the subsites�1,þ1, andtheir surroundings in the WT (Table 1). The O3H is hydrogenbonded to Oδ1(Asp214) and remains throughout the whole glycosy-lation process. Nε2 of His228, located around subsite þ1, acts as ahydrogen bond donor to the Asp214 carboxyl group, which formsanother hydrogen bond with the O3H group in subsite þ1. Thesetwo hydrogen bonds, unchanged during the proton transfer andnucleophilic attack processes, effectively restrain the positionofHis228

with the assist of a third strengthening hydrogen bond, OγHγ

(Ser174) 3 3 3Nδ1(His228). Oγ(Ser174) is a hydrogen bond acceptor

to the Nε2Hε22 group of residue Gln175 which is also hydrogenbonded toO2

0 as a donor, while NH(Ser174) forms a hydrogen bondto Oε1(Glu212) in the ES of WT (2.90 Å). The NH(Ser174) 3 3 3O

ε1-(Glu212) hydrogen bond weakens during the reaction, especiallyalong the covalent C1

0�Oε2(Glu212) bond formation in the nucleo-philic attack process. All these H-bonds can be observed in mutantsexcept that the Nε2H(His228)�Oδ1(Asp214) hydrogen bond disap-pears in the D214N mutant, and NH(Ser174) 3 3 3O

ε1(Glu212) dis-appears during the nucleophilic attack reaction in D214N and doesnot exist at all inE212Q(Tables 1, 2, and3). In addition,Oε1(Gln175)couples bidentately with the Nη1�Hη12 and Nη2�Hη22 groups ofArg251 through a hydrogen bond which only exists in the WT andD214N mutant.In the WT, the distance between two carboxylate groups of the

donor residue (Glu217) and the nucleophile (Glu212) is about 6.0(0.5 Å, in good agreement with the commonly observed results inprevious experimental and theoretical investigations.4,10,11,18,19 Thisdistance, in the E212Q mutant, lengthens from 7.0 to 9.0 Åfollowing the proton transfer and then is reduced back to ∼7.0 Åduring the nucleophilic attack process. The distance change of thesetwo carboxylate groupsmay be related to the high free energy barrierduring the nucleophilic attack process. In D214N, the distancebetween two carboxylate groups of Glu212 and Glu217 is around 7.5( 0.5 Å, longer than that inWT, but shorter than that in E212Q onaverage.At the beginning of the proton transfer, the ring in subsite�1

(the ring labeled in Scheme 1) is in chair conformation and tiltsby 60� with the plane defined by the other three parallelneighboring rings (subsites �2, þ1, and þ2). As the reactionproceeds, the tilt angle decreases rapidly. The glycosyl ring insubsite�1 varies from chair (4C1) to skew-boat (

1S3) conforma-tion, through a half-chair (4H3) transition state during the protontransfer reaction. These phenomena are shared by the WT andthe mutants. The corresponding IM configurations are shown inFigure 4, and a common characteristic is observed: the C1, C2,C5, and O5 atoms form a plane. This is in good agreement withthe previous reports.7,17 This ring conformation changes back to4C1 after the nucleophilic attack for WT.18 However, a 2,5Bconformation comes into being in the GI state for the E212Q andD214N mutants.3.2.3. Binding Subsite þ2. The Nε(Arg251) is hydrogen

bonded with the carboxylate group of the Asp259 residue in threeES forms. During the whole reaction, the variation of thishydrogen bond is small in the WT and D214N mutant. However,the change is substantial during the nucleophilic attack step inE212Q. Arg394, located at the cellobiose exit, interacts with O1, O5,and O6 of subsite þ2. In the ES, O1 locates between two NH2

groups of Arg394, while the O5 and O6 are positioned inside thechannel. During the proton transfer and the nucleophilic attackprocesses, cellobiose approaches toward Arg394 and moves out ofthe active site. Further study will be needed to prove whether theinteraction between the two facilitates the departure of cellobiose.

4. CONCLUSIONS

On the basis of the QM/MD simulation results, the config-urations and the coupling details around the enzyme active siteare analyzed. Along with the transfer of a proton from the donorresidue, Glu217, to the glycosidic O4, the covalent CO bond,O4�C1

0, is broken, and the cellobiose departs from the activesite. At the same time, the distance between C1

0 and the

Figure 4. Snapshots of the enzyme�substrate intermediate. The con-figurations of residues 212, 214, and 217 as well as the ring conforma-tions of subunit �1 are shown.



nucleophilic groupGlu212 (or Gln212) decreases, which facilitatesthe subsequent nucleophilic attack reaction. A detailed mecha-nism is presented to illustrate the decrease of activity of themutants during the hydrolysis of cellulose. The dramatic loss ofenzyme catalytic capability of the E212Q mutant is due to theoverall activation barrier increase of ∼4.0 kcal/mol. In contrast,the D214N mutation only leads to an increase of the activationenergy by∼0.4 kcal/mol, which accounts for the slight decreaseof the catalytic efficiency of this mutant. The detailed geometricanalysis suggests that a hydrogen bond network contributes tothe active site interaction and enzymatic catalytic process. Thedistance of the two carboxylate groups of Glu212 and Glu217 islengthened for the mutations.

’AUTHOR INFORMATION

Corresponding Author*Tel.: þ86 532 80662792. Fax: þ86 532 80662778. E-mail:[email protected].

’ACKNOWLEDGMENT

We are thankful to Supercomputing Center of ChineseAcademy of Sciences (CAS) for providing the computer re-sources and time. This work was supported by 100 TalentProject, the Knowledge Innovation Program of the CAS(Grant No. KSCX2-EW-J-10), the Director Innovation Founda-tion of Qingdao Institute of Biomass Energy and BioprocessTechnology of CAS, and the Foundation for Outstanding YoungScientist in Shandong Province (No. BS2010NJ020).

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mutational effects on the catalytic mechanism of cellobiohydrolase i from trichoderma...

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