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• W2084828551 abstract "Cellobiohydrolases are exo-active glycosyl hydrolases that processively convert cellulose to soluble sugars, typically cellobiose. They effectively break down crystalline cellulose and make up a major component in industrial enzyme mixtures used for deconstruction of lignocellulosic biomass. Identification of the rate-limiting step for cellobiohydrolases remains controversial, and recent reports have alternately suggested either association (on-rate) or dissociation (off-rate) as the overall bottleneck. Obviously, this uncertainty hampers both fundamental mechanistic understanding and rational design of enzymes with improved industrial applicability. To elucidate the role of on- and off-rates, respectively, on the overall kinetics, we have expressed a variant in which a tryptophan residue (Trp-38) in the middle of the active tunnel has been replaced with an alanine. This mutation weakens complex formation, and the population of substrate-bound W38A was only about half of the wild type. Nevertheless, the maximal, steady-state rate was twice as high for the variant enzyme. It is argued that these opposite effects on binding and activity can be reconciled if the rate-limiting step is after the catalysis (i.e. in the dissociation process). Cellobiohydrolases are exo-active glycosyl hydrolases that processively convert cellulose to soluble sugars, typically cellobiose. They effectively break down crystalline cellulose and make up a major component in industrial enzyme mixtures used for deconstruction of lignocellulosic biomass. Identification of the rate-limiting step for cellobiohydrolases remains controversial, and recent reports have alternately suggested either association (on-rate) or dissociation (off-rate) as the overall bottleneck. Obviously, this uncertainty hampers both fundamental mechanistic understanding and rational design of enzymes with improved industrial applicability. To elucidate the role of on- and off-rates, respectively, on the overall kinetics, we have expressed a variant in which a tryptophan residue (Trp-38) in the middle of the active tunnel has been replaced with an alanine. This mutation weakens complex formation, and the population of substrate-bound W38A was only about half of the wild type. Nevertheless, the maximal, steady-state rate was twice as high for the variant enzyme. It is argued that these opposite effects on binding and activity can be reconciled if the rate-limiting step is after the catalysis (i.e. in the dissociation process). Enzymatic breakdown of crystalline cellulose has proven challenging to describe in molecular detail. This is probably the result of a complex enzyme architecture with different domains and moieties that interacts with the heterogeneous surface of the insoluble substrate (1Teeri T.T. Crystalline cellulose degradation: new insight into the function of cellobiohydrolases.Trends Biotechnol. 1997; 15: 160-167Abstract Full Text PDF Scopus (532) Google Scholar, 2Zhang Y.H. Lynd L.R. Toward an aggregated understanding of enzymatic hydrolysis of cellulose: noncomplexed cellulase systems.Biotechnol. Bioeng. 2004; 88: 797-824Crossref PubMed Scopus (1489) Google Scholar, 3Bansal P. Hall M. Realff M.J. Lee J.H. Bommarius A.S. Modeling cellulase kinetics on lignocellulosic substrates.Biotechnol. Adv. 2009; 27: 833-848Crossref PubMed Scopus (335) Google Scholar, 4Beckham G.T. Ståhlberg J. Knott B.C. Himmel M.E. Crowley M.F. Sandgren M. Sørlie M. Payne C.M. Towards a molecular-level theory of carbohydrate processivity in glycoside hydrolases.Curr. Opin. Biotechnol. 2014; 27: 96-106Crossref PubMed Scopus (75) Google Scholar). Moreover, the linear β-1,4-glucan chains in the highly condensed substrate are connected by strong intermolecular forces, which compete with enzyme-substrate interactions during the hydrolysis and hence add to the complexity of the process (5Beckham G.T. Matthews J.F. Peters B. Bomble Y.J. Himmel M.E. Crowley M.F. Molecular-level origins of biomass recalcitrance: decrystallization free energies for four common cellulose polymorphs.J. Phys. Chem. B. 2011; 115: 4118-4127Crossref PubMed Scopus (161) Google Scholar, 6Payne C.M. Jiang W. Shirts M.R. Himmel M.E. Crowley M.F. Beckham G.T. Glycoside hydrolase processivity is directly related to oligosaccharide binding free energy.J. Am. Chem. Soc. 2013; 135: 18831-18839Crossref PubMed Scopus (72) Google Scholar). The most extensively studied cellulase is the cellobiohydrolase Cel7A from Trichoderma reesei (an anamorph of the fungus Hypocrea jecorina). TrCel7A is a multidomain enzyme comprising a catalytic domain (core), which is connected by a highly glycosylated linker peptide to a family 1 carbohydrate-binding module (CBM1) 3The abbreviations used are: CBMcarbohydrate-binding moduleBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. (7Abuja P.M. Schmuck M. Pilz I. Tomme P. Claeyssens M. Esterbauer H. Structural and functional domains of cellobiohydrolase I from Trichoderma reesei.Eur. Biophys. J. 1988; 15: 339-342Crossref Scopus (117) Google Scholar). The three-dimensional crystal structure of the catalytic domain in complex with different cello-oligosaccharides has been solved, revealing a 50-Å-long active site tunnel with 10 glucosyl-binding sites, numbered from −7 at the entrance of the tunnel to +3 at the exit (8Divne C. Ståhlberg J. Teeri T.T. Jones T.A. High-resolution crystal structures reveal how a cellulose chain is bound in the 50 Å long tunnel of cellobiohydrolase I from Trichoderma reesei.J. Mol. Biol. 1998; 275: 309-325Crossref PubMed Scopus (363) Google Scholar). The enzyme hydrolyzes cellulose processively from the reducing end with the scissile bond between subsite −1 and +1, hence leaving cellobiose in the +1,+2 expulsion site. Analysis of processive hydrolysis requires consideration of a number of steps, including adsorption, surface diffusion, recognition, “threading,” complex formation, catalysis, product expulsion, processive sliding, and dissociation (4Beckham G.T. Ståhlberg J. Knott B.C. Himmel M.E. Crowley M.F. Sandgren M. Sørlie M. Payne C.M. Towards a molecular-level theory of carbohydrate processivity in glycoside hydrolases.Curr. Opin. Biotechnol. 2014; 27: 96-106Crossref PubMed Scopus (75) Google Scholar, 9Chundawat S.P.S. Beckham G.T. Himmel M.E. Dale B.E. Deconstruction of lignocellulosic biomass to fuels and chemicals.Annu. Rev. Chem. Biomol. Eng. 2011; 2: 121-145Crossref PubMed Scopus (740) Google Scholar). Many of these steps remain poorly understood partly because their isolation has proven difficult in experiments (4Beckham G.T. Ståhlberg J. Knott B.C. Himmel M.E. Crowley M.F. Sandgren M. Sørlie M. Payne C.M. Towards a molecular-level theory of carbohydrate processivity in glycoside hydrolases.Curr. Opin. Biotechnol. 2014; 27: 96-106Crossref PubMed Scopus (75) Google Scholar) and partly as a result of the heterogeneous environment in which these enzyme work (2Zhang Y.H. Lynd L.R. Toward an aggregated understanding of enzymatic hydrolysis of cellulose: noncomplexed cellulase systems.Biotechnol. Bioeng. 2004; 88: 797-824Crossref PubMed Scopus (1489) Google Scholar, 10Himmel M.E. Ding S.Y. Johnson D.K. Adney W.S. Nimlos M.R. Brady J.W. Foust T.D. Biomass recalcitrance: engineering plants and enzymes for biofuels production.Science. 2007; 315: 804-807Crossref PubMed Scopus (3445) Google Scholar). carbohydrate-binding module 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. It is of particular importance to identify the slowest and hence rate-determining step in the catalytic cycle. Numerous earlier studies have addressed this question, and interestingly, it appears as if the conclusions fall mainly into two mutually conflicting groups. Thus, a number of works have used either direct experimental evidence or re-examination of broader, previously published material to conclude that the overall rate is governed by the association of enzyme and substrate, particularly the placement of a cellulose strand in the long active tunnel (11Shang B.Z. Chang R. Chu J.W. Systems-level modeling with molecular resolution elucidates the rate-limiting mechanisms of cellulose decomposition by cellobiohydrolases.J. Biol. Chem. 2013; 288: 29081-29089Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 12Fox J.M. Jess P. Jambusaria R.B. Moo G.M. Liphardt J. Clark D.S. Blanch H.W. A single-molecule analysis reveals morphological targets for cellulase synergy.Nat. Chem. Biol. 2013; 9: 356-361Crossref PubMed Scopus (55) Google Scholar, 13von Ossowski I. Ståhlberg J. Koivula A. Piens K. Becker D. Boer H. Harle R. Harris M. Divne C. Mahdi S. Zhao Y. Driguez H. Claeyssens M. Sinnott M.L. Teeri T.T. Engineering the exo-loop of Trichoderma reesei cellobiohydrolase, Cel7A. A comparison with Phanerochaete chrysosporium Cel7D.J. Mol. Biol. 2003; 333: 817-829Crossref PubMed Scopus (136) Google Scholar, 14Wilson D.B. Cellulases and biofuels.Curr. Opin. Biotechnol. 2009; 20: 295-299Crossref PubMed Scopus (348) Google Scholar, 15Nakamura A. Watanabe H. Ishida T. Uchihashi T. Wada M. Ando T. Igarashi K. Samejima M. Trade-off between processivity and hydrolytic velocity of cellobiohydrolases at the surface of crystalline cellulose.J. Am. Chem. Soc. 2014; 136: 4584-4592Crossref PubMed Scopus (59) Google Scholar, 16Nakamura A. Tsukada T. Auer S. Furuta T. Wada M. Koivula A. Igarashi K. Samejima M. The tryptophan residue at the active site tunnel entrance of Trichoderma reesei cellobiohydrolase Cel7A is important for initiation of degradation of crystalline cellulose.J. Biol. Chem. 2013; 288: 13503-13510Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 17Fox J.M. Levine S.E. Clark D.S. Blanch H.W. Initial- and processive-cut products reveal cellobiohydrolase rate limitations and the role of companion enzymes.Biochemistry. 2012; 51: 442-452Crossref PubMed Scopus (87) Google Scholar, 18Maurer S.A. Bedbrook C.N. Radke C.J. Cellulase adsorption and reactivity on a cellulose surface from flow ellipsometry.Ind. Eng. Chem. Res. 2012; 51: 11389-11400Crossref Scopus (47) Google Scholar). In contrast to this, other studies, including work by this group on the pre-steady state kinetics of wild type Cel7A, have suggested that the formation of the activated complex is quite rapid and that dissociation of enzyme, which is in a position where further processive movement is hindered, is the rate-limiting step (19Kurasin M. Väljamäe P. Processivity of cellobiohydrolases is limited by the substrate.J. Biol. Chem. 2011; 286: 169-177Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 20Jalak J. Väljamäe P. Mechanism of initial rapid rate retardation in cellobiohydrolase catalyzed cellulose hydrolysis.Biotechnol. Bioeng. 2010; 106: 871-883Crossref PubMed Scopus (118) Google Scholar, 21Jalak J. Kurašin M. Teugjas H. Väljamäe P. Endo-exo synergism in cellulose hydrolysis revisited.J. Biol. Chem. 2012; 287: 28802-28815Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 22Cruys-Bagger N. Elmerdahl J. Praestgaard E. Tatsumi H. Spodsberg N. Borch K. Westh P. Pre-steady-state kinetics for hydrolysis of insoluble cellulose by cellobiohydrolase Cel7A.J. Biol. Chem. 2012; 287: 18451-18458Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 23Cruys-Bagger N. Elmerdahl J. Praestgaard E. Borch K. Westh P. A steady-state theory for processive cellulases.FEBS J. 2013; 280: 3952-3961Crossref PubMed Scopus (44) Google Scholar, 24Cruys-Bagger N. Tatsumi H. Ren G.R. Borch K. Westh P. Transient kinetics and rate-limiting steps for the processive cellobiohydrolase Cel7A: effects of substrate structure and carbohydrate binding domain.Biochemistry. 2013; 52: 8938-8948Crossref PubMed Scopus (63) Google Scholar, 25Cruys-Bagger N. Tatsumi H. Borch K. Westh P. A graphene screen-printed carbon electrode for real-time measurements of unoccupied active sites in a cellulase.Anal. Biochem. 2014; 447: 162-168Crossref PubMed Scopus (18) Google Scholar). To elucidate this question, we have designed, expressed, and purified variants of TrCel7A with lower affinity for the substrate. The idea was that the kinetic behavior of an enzyme with a less stable substrate complex could reveal whether the bottleneck was located before or after the activated complex (i.e. whether rate limitation was one of the associating or dissociating steps). Specifically, we replaced Trp-38 located in subsite −4 (Fig. 1) in the active tunnel with Ala. Previous work has suggested that this tryptophan residue interacts with the substrate (8Divne C. Ståhlberg J. Teeri T.T. Jones T.A. High-resolution crystal structures reveal how a cellulose chain is bound in the 50 Å long tunnel of cellobiohydrolase I from Trichoderma reesei.J. Mol. Biol. 1998; 275: 309-325Crossref PubMed Scopus (363) Google Scholar, 26Taylor C.B. Payne C.M. Himmel M.E. Crowley M.F. McCabe C. Beckham G.T. Binding site dynamics and aromatic-carbohydrate interactions in processive and non-processive family 7 glycoside hydrolases.J. Phys. Chem. B. 2013; 117: 4924-4933Crossref PubMed Scopus (50) Google Scholar), and its replacement with a non-aromatic residue was expected to weaken the complex. Here, we report a comparative analysis of steady-state kinetics, adsorption, and processivity for wild type TrCel7A and the Trp-38 → Ala variant, henceforth termed WT and W38A, respectively. To assess possible effects of the CBM, we have made this comparison both for the intact enzyme and the catalytic domain alone. All experiments were conducted in a standard 50 mm acetate buffer, 2 mm CaCl2, pH 5.0. All enzymes were expressed heterologously in Aspergillus oryzae and purified as described elsewhere (27Westh, P., Kari, J., Olsen, J., Borch, K., Jensen, K., Krogh, K. B. R. M., (May 1, 2014) PCT International Patent Application WO/2014/064115, C12N 9/00 Ed.Google Scholar). Core variants (i.e. the catalytic domain alone) were made by introducing a BamHI restriction-site in the TrCel7A gene at the end of the core domain, making it possible to splice out the core domain from the TrCel7A plasmid or W38A plasmid. All purified enzyme stocks showed a single band in SDS NuPAGE 4–12% BisTris gel (GE Healthcare), and the absence of specific contamination by β-glucosidase was confirmed by the lack of activity against p-nitrophenyl β-d-glucopyranoside. Henceforth, we will use subscript “intact” for enzymes with and “core” for enzymes without the CBM. Enzyme concentrations were determined by absorbance at 280 nm using molar extinction coefficients of 84,810 m−1 cm−1, 79,120 m−1 cm−1, 79,210 m−1 cm−1, and 73,520 m−1 cm−1 for WTintact, W38Aintact, WTcore, and W38Acore, respectively. The cellobiose production was monitored by an amperometric enzyme biosensor based on cellobiose dehydrogenase from Phanerochaete chrysosporium adsorbed onto the surface of a benzoquinone-modified carbon paste electrode as described in detail previously (28Cruys-Bagger N. Ren G. Tatsumi H. Baumann M.J. Spodsberg N. Andersen H.D. Gorton L. Borch K. Westh P. An amperometric enzyme biosensor for real-time measurements of cellobiohydrolase activity on insoluble cellulose.Biotechnol. Bioeng. 2012; 109: 3199-3204Crossref PubMed Scopus (38) Google Scholar). Measurements were carried out at 25 °C in a 5-ml water-jacketed glass cell connected to a water bath. Avicel PH 101 (Sigma-Aldrich) suspensions were stirred at 600 rpm, and enzyme was injected to a final concentration of 100 nm from a Chemyx Fusion 100 syringe pump with an injection time of 1.0 s. All measurements were made in duplicate with calibration before and after each run, using the average sensitivity as the calibration factor (28Cruys-Bagger N. Ren G. Tatsumi H. Baumann M.J. Spodsberg N. Andersen H.D. Gorton L. Borch K. Westh P. An amperometric enzyme biosensor for real-time measurements of cellobiohydrolase activity on insoluble cellulose.Biotechnol. Bioeng. 2012; 109: 3199-3204Crossref PubMed Scopus (38) Google Scholar). The adsorption of enzyme on Avicel was measured in 96-well microtiter plates by adding substrate and enzyme to a final volume of 300 μl in each well. The substrate load was 10 g/liter for enzymes with CBM and 30 g/liter for the core variants, and the enzyme concentration was varied from 0 to 5 μm. The plate was sealed and incubated for 1 h at 25 °C and 1200 rpm orbital mixing. After incubation, the plate was centrifuged at 3500 rpm for 5 min, and 100 μl of supernatant was transferred to black 96-well microtiter plates. Intrinsic protein fluorescence was determined by measuring the emission at 340 nm at an excitation wavelength of 280 nm. In all experiments, an enzyme standard curve was made using the enzyme in question as reference. The production of glucose, cellobiose, and cellotriose was measured at different time points by taking 500-μl subsets of a 50-ml reaction mixture, containing 10 g/liter Avicel and 0.50 μm enzyme at 25.0 °C. Subsets were quenched by mixing with the same volume 0.1 m NaOH, and following centrifugation, the concentrations of glucose, cellobiose, and cellotriose in the supernatants were measured on a Dionex ICS-5000 ion chromatograph (Thermo Fisher Scientific, Waltham, MA) as described elsewhere (27Westh, P., Kari, J., Olsen, J., Borch, K., Jensen, K., Krogh, K. B. R. M., (May 1, 2014) PCT International Patent Application WO/2014/064115, C12N 9/00 Ed.Google Scholar, 29Alasepp K. Borch K. Cruys-Bagger N. Badino S. Jensen K. Sørensen T.H. Windahl M.S. Westh P. In situ stability of substrate associated cellulases studied by scanning calorimetry.Langmuir. 2014; 30: 7134-7142Crossref PubMed Scopus (15) Google Scholar). Fig. 2 shows real-time data obtained by the P. chrysosporium cellobiose dehydrogenase biosensor. The two top panels illustrate the cellobiose production for the enzymes with CBM, WTintact, and W38Aintact. Analogously, the bottom panels present data for the two core variants, WTcore and W38Acore. The enzyme concentration was 100 nm in all trials, whereas the load of Avicel was varied over 2 orders of magnitude (0.5–60 g/liter). The results show that enzymes with the W38A mutation produce more cellobiose than the corresponding wild type except at very low Avicel loads. We have previously suggested (23Cruys-Bagger N. Elmerdahl J. Praestgaard E. Borch K. Westh P. A steady-state theory for processive cellulases.FEBS J. 2013; 280: 3952-3961Crossref PubMed Scopus (44) Google Scholar, 30Praestgaard E. Elmerdahl J. Murphy L. Nymand S. McFarland K.C. Borch K. Westh P. A kinetic model for the burst phase of processive cellulases.FEBS J. 2011; 278: 1547-1560Crossref PubMed Scopus (80) Google Scholar) that experimental data for processive enzymes may be analyzed by a reaction scheme (Scheme 1), according to which the free enzyme, E, combines with a cellulose strand, Cm, to form an activated complex, ECm. The enzyme subsequently moves along the strand, which is sequentially shortened by one cellobiose, moiety C, for each step (so the strand is converted to Cm-1, Cm-2, etc.). In this process, the activated complexes (ECm-1, ECm-2 …) are all allowed two reaction pathways. They may either go through another catalytic cycle to produce a cellobiose and a correspondingly shortened cellulose strand, or they may dissociate. This reaction mechanism can be kinetically described by four parameters. These are the rate constants for association (kon), catalysis (kcat), and dissociation (koff) and the processivity number, n (the average number of sequential steps made following the attack of one cellulose strand). At quasi-steady state, the rate of cellobiose production according to the processive reaction in Scheme 1, pVSS, attains the usual hyperbolic form (23Cruys-Bagger N. Elmerdahl J. Praestgaard E. Borch K. Westh P. A steady-state theory for processive cellulases.FEBS J. 2013; 280: 3952-3961Crossref PubMed Scopus (44) Google Scholar), pVSS= pVmaxS pKm+S(Eq. 1) where S is the load of substrate in g/liter, and the processive kinetic parameters, pKm and pVmax, are defined by the processivity, n, and the rate constants from Scheme 1. pKm=koffkon(Eq. 2) and pVmax=βE0kcat(Eq. 3) (Eq. 1), (Eq. 2), (Eq. 3) rely on the assumption that the substrate is in excess (i.e. that the concentration of attack sites for enzyme on the substrate surface is much larger than the enzyme concentration). This has previously been shown to be a reasonable assumption for conditions similar to those used here (22Cruys-Bagger N. Elmerdahl J. Praestgaard E. Tatsumi H. Spodsberg N. Borch K. Westh P. Pre-steady-state kinetics for hydrolysis of insoluble cellulose by cellobiohydrolase Cel7A.J. Biol. Chem. 2012; 287: 18451-18458Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 23Cruys-Bagger N. Elmerdahl J. Praestgaard E. Borch K. Westh P. A steady-state theory for processive cellulases.FEBS J. 2013; 280: 3952-3961Crossref PubMed Scopus (44) Google Scholar). We note that the main difference between the parameters for processive enzyme activity given in Equations 2 and 3 and the analogous parameters in the conventional Michaelis-Menten equation is the constant, β. This so-called kinetic processivity coefficient is defined, β = 1 − (kcat/(kcat + koff))n and may be interpreted as the probability that the enzyme will dissociate from the cellulose strand before completing the average number (n) of processive steps (23Cruys-Bagger N. Elmerdahl J. Praestgaard E. Borch K. Westh P. A steady-state theory for processive cellulases.FEBS J. 2013; 280: 3952-3961Crossref PubMed Scopus (44) Google Scholar). The experimental data in Fig. 2 are typical for cellulases in the sense that a truly constant reaction rate (i.e. a fully linear progress curve) is never observed. As argued previously (23Cruys-Bagger N. Elmerdahl J. Praestgaard E. Borch K. Westh P. A steady-state theory for processive cellulases.FEBS J. 2013; 280: 3952-3961Crossref PubMed Scopus (44) Google Scholar), this requires some compromise in the definition of the steady-state rate, and it was recommended to assign pVSS as the slope of the concentration trace after the initial burst, which represents a non-steady-state condition (23Cruys-Bagger N. Elmerdahl J. Praestgaard E. Borch K. Westh P. A steady-state theory for processive cellulases.FEBS J. 2013; 280: 3952-3961Crossref PubMed Scopus (44) Google Scholar, 31Kipper K. Väljamäe P. Johansson G. Processive action of cellobiohydrolase Cel7A from Trichoderma reesei is revealed as “burst” kinetics on fluorescent polymeric model substrates.Biochem. J. 2005; 385: 527-535Crossref PubMed Scopus (96) Google Scholar) but before the rate has been significantly reduced by other (as of yet partially unknown) factors. In accordance with this, we fitted a line to the data from 100 to 150 s in Fig. 2 and used the slope as a measure of pVSS. Results are plotted as a function of the Avicel load in Fig. 3. Comparisons of data for the two enzymes with CBM (WTintact and W38Aintact) and the two catalytic domains (WTcore and W38Acore), respectively, again show that the W38A mutation slightly decreases activity at very low substrate load but significantly promotes pVSS when the Avicel load approaches saturating levels. Fig. 3 also shows that Equation 1 accounts well for the experimental data for all four enzymes, and the kinetic parameters derived from the regression analysis based on Equation 1 are listed in Table 1. These parameters reveal that the two W38A variants have 1.6–2 times higher pVmax compared with the corresponding enzymes without this mutation. Conversely, the affinity for the substrate is lowered by the W38A mutation (pKm is increased 4 times for the intact enzyme and 2 times for the core variant).TABLE 1Steady-state kinetic and adsorption parameters for the four investigated TrCel7A variants, WTintact, W38Aintact, WTcore, and W38AcoreEnzymeKinetic parametersAdsorption parameterspVmaxpKmpVmax/pKmΓmaxKdΓmax/Kdnm/sg/liternmol/s/gnmol/gμmliter/gWTintact19.5 ±0.61.54 ± 0.2312.7 ± 1.93195 ± 70.145 ± 0.0191.345 ± 0.183W38Aintact38.4 ± 0.75.95 ± 0.346.5 ± 0.39114 ± 40.544 ± 0.0610.210 ± 0.025WTcore34.4 ± 1.812.6 ± 1.822.7 ± 0.4230 ± 21.43 ± 0.1950.021 ± 0.003W38Acore53.2 ± 2.924.3 ± 2.872.2 ± 0.2822 ± 32.97 ± 0.6200.008 ± 0.002 Open table in a new tab The effect of the W38A mutation on substrate affinity was further investigated by adsorption measurements. Results in Fig. 4 show the characteristic adsorption saturation and a strong effect of the CBM, which have been discussed in detail earlier both for TrCel7A and numerous related cellulases (32Ståhlberg J. Johansson G. Pettersson G. A new model for enzymatic hydrolysis of cellulose based on the two-domain structure of cellobiohydrolase I.Nat. Biotechnol. 1991; 9: 286-290Crossref Scopus (90) Google Scholar, 33Wahlström R. Rahikainen J. Kruus K. Cellulose hydrolysis and binding with Trichoderma reesei Cel5A and Cel7A and their core domains in ionic liquid solutions.Biotechnol. Bioeng. 2014; 111: 726-733Crossref PubMed Scopus (24) Google Scholar, 34Igarashi K. Wada M. Hori R. Samejima M. Surface density of cellobiohydrolase on crystalline celluloses.FEBS J. 2006; 273: 2869-2878Crossref PubMed Scopus (65) Google Scholar). In the current context, it is more important to note that the W38A mutation significantly reduces binding both in the intact enzyme and in the core variant. To quantify this, we analyzed the data with respect to a standard Langmuir isotherm, Γ, where the variables E and Γ represent free concentration and adsorbed amount of enzyme, respectively, and Γmax and Kd are the usual Langmuir parameters (saturation coverage and dissociation constant). The Langmuir isotherm was found to account well for the experimental data, and the parameters are listed in Table 1. It appears that the mutation weakens the interaction significantly because Kd increased 3.7 times in the intact enzyme and 2.1 times in the core variant. The saturation coverage, Γmax, was also reduced (30–40%) by the mutation, indicating a reduced substrate accessibility for the mutant. Progress curves for glucose, cellobiose, and cellotriose produced by WTintact and W38Aintact are shown in Fig. 5. These measurements were made to assess the influence of the W38A mutation on the processivity. It has previously been suggested that processivity can be estimated from the cellobiose/cellotriose ratio of either concentrations or steady-state production rates (31Kipper K. Väljamäe P. Johansson G. Processive action of cellobiohydrolase Cel7A from Trichoderma reesei is revealed as “burst” kinetics on fluorescent polymeric model substrates.Biochem. J. 2005; 385: 527-535Crossref PubMed Scopus (96) Google Scholar, 35Horn S.J. Sørlie M. Vårum K.M. Väljamäe P. Eijsink V.G. Measuring processivity.Methods Enzymol. 2012; 510: 69-95Crossref PubMed Scopus (77) Google Scholar, 36Vuong T.V. Wilson D.B. Processivity, synergism, and substrate specificity of Thermobifida fusca Cel6B.Appl. Environ. Microbiol. 2009; 75: 6655-6661Crossref PubMed Scopus (66) Google Scholar). Applying these principles, we found processivities of 23–24 for WTintact and 15–16 for W38Aintact. Product profiles were only measured for one enzyme concentration, and the following discussion of processivity hence neglects a possible dependence of n on the enzyme/substrate ratio. We note, however, that the enzyme/substrate ratio in the product profile measurements (Fig. 5) was comparable with the ratio in the kinetic measurements (Fig. 2) for substrate loads around pKm and hence that the two types of experiments appear compatible. The processivity of the wild type TrCel7A on Avicel has been measured before (15Nakamura A. Watanabe H. Ishida T. Uchihashi T. Wada M. Ando T. Igarashi K. Samejima M. Trade-off between processivity and hydrolytic velocity of cellobiohydrolases at the surface of crystalline cellulose.J. Am. Chem. Soc. 2014; 136: 4584-4592Crossref PubMed Scopus (59) Google Scholar, 22Cruys-Bagger N. Elmerdahl J. Praestgaard E. Tatsumi H. Spodsberg N. Borch K. Westh P. Pre-steady-state kinetics for hydrolysis of insoluble cellulose by cellobiohydrolase Cel7A.J. Biol. Chem. 2012; 287: 18451-18458Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 31Kipper K. Väljamäe P. Johansson G. Processive action of cellobiohydrolase Cel7A from Trichoderma reesei is revealed as “burst” kinetics on fluorescent polymeric model substrates.Biochem. J. 2005; 385: 527-535Crossref PubMed Scopus (96) Google Scholar), and the current result is in line with these earlier reports. We note that relationships between product profiles and processivity have been extensively discussed (4Beckham G.T. Ståhlberg J. Knott B.C. Himmel M.E. Crowley M.F. Sandgren M. Sørlie M. Payne C.M. Towards a molecular-level theory of carbohydrate processivity in glycoside hydrolases.Curr. Opin. Biotechnol. 2014; 27: 96-106Crossref PubMed Scopus (75) Google Scholar, 35Horn S.J. Sørlie M. Vårum K.M. Väljamäe P. Eijsink V.G. Measuring processivity.Methods Enzymol. 2012; 510: 69-95Crossref PubMed Scopus (77) Google Scholar, 37Wilson D.B. Kostylev M. Cellulase processivity.Methods Mol. Biol. 2012; 908: 93-99PubMed Google Scholar), and somewhat deviant principles for the calculation of n have been put forward (22Cruys-Bagger N. Elmerdahl J. Praestgaard E. Tatsumi H. Spodsberg N. Borch K. Westh P. Pre-steady-state kinetics for hydrolysis of insoluble cellulose by cellobiohydrolase Cel7A.J. Biol. Chem. 2012; 287: 18451-18458Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 31Kipper K. Väljamäe P. Johansson G. Processive action of cellobiohydrolase Cel7A from Trichoderma reesei is revealed as “burst” kinetics on fluorescent polymeric model substrates.Biochem. J. 2005; 385: 527-535Crossref PubMed Scopus (96) Google Scholar, 36Vuong T.V. Wilson D.B. Processivity, synergism, and substrate" @default.
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• W2084828551 title "Kinetics of Cellobiohydrolase (Cel7A) Variants with Lowered Substrate Affinity" @default.
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