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• W2012194120 abstract "A β-glucosidase, designated isoenzyme βII, from germinated barley (Hordeum vulgare L.) hydrolyzes aryl-β-glucosides and shares a high level of amino acid sequence similarity with β-glucosidases of diverse origin. It releases glucose from the non-reducing termini of cellodextrins with catalytic efficiency factors,k cat/K m, that increase approximately 9-fold as the degree of polymerization of these substrates increases from 2 to 6. Thus, the enzyme has a specificity and action pattern characteristic of both β-glucosidases (EC3.2.1.21) and the polysaccharide exohydrolase, (1,4)-β-glucan glucohydrolase (EC 3.2.1.74). At high concentrations (100 mm) of 4-nitrophenyl β-glucoside, β-glucosidase isoenzyme βII catalyzes glycosyl transfer reactions, which generate 4-nitrophenyl-β-laminaribioside, -cellobioside, and -gentiobioside. Subsite mapping with cellooligosaccharides indicates that the barley β-glucosidase isoenzyme βII has six substrate-binding subsites, each of which binds an individual β-glucosyl residue. Amino acid residues Glu181 and Glu391 are identified as the probable catalytic acid and catalytic nucleophile, respectively. The enzyme is a family 1 glycoside hydrolase that is likely to adopt a (β/α)8 barrel fold and in which the catalytic amino acid residues appear to be located at the bottom of a funnel-shaped pocket in the enzyme. A β-glucosidase, designated isoenzyme βII, from germinated barley (Hordeum vulgare L.) hydrolyzes aryl-β-glucosides and shares a high level of amino acid sequence similarity with β-glucosidases of diverse origin. It releases glucose from the non-reducing termini of cellodextrins with catalytic efficiency factors,k cat/K m, that increase approximately 9-fold as the degree of polymerization of these substrates increases from 2 to 6. Thus, the enzyme has a specificity and action pattern characteristic of both β-glucosidases (EC3.2.1.21) and the polysaccharide exohydrolase, (1,4)-β-glucan glucohydrolase (EC 3.2.1.74). At high concentrations (100 mm) of 4-nitrophenyl β-glucoside, β-glucosidase isoenzyme βII catalyzes glycosyl transfer reactions, which generate 4-nitrophenyl-β-laminaribioside, -cellobioside, and -gentiobioside. Subsite mapping with cellooligosaccharides indicates that the barley β-glucosidase isoenzyme βII has six substrate-binding subsites, each of which binds an individual β-glucosyl residue. Amino acid residues Glu181 and Glu391 are identified as the probable catalytic acid and catalytic nucleophile, respectively. The enzyme is a family 1 glycoside hydrolase that is likely to adopt a (β/α)8 barrel fold and in which the catalytic amino acid residues appear to be located at the bottom of a funnel-shaped pocket in the enzyme. Two β-glucosidases of apparent molecular mass 62,000 have been purified from extracts of germinated barley grain (1Simos G. Panagiotidis C.A. Skoumbas A. Chali D. Ouzounis C. Georgatsos J.G. Biochim. Biophys. Acta. 1994; 1199: 52-58Crossref PubMed Scopus (23) Google Scholar, 2Leah R. Kigel J. Svendsen I. Mundy J. J. Biol. Chem. 1995; 270: 15789-15797Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 3Hrmova M. Harvey A.J. Wang J. Shirley N.J. Jones G.P. Stone B.A. Høj P.B. Fincher G.B. J. Biol. Chem. 1996; 271: 5277-5286Abstract Full Text PDF PubMed Google Scholar) and can be classified in the family 1 group of glycosyl hydrolases and related enzymes (4Henrissat B. Bairoch A. Biochem. J. 1993; 293: 781-788Crossref PubMed Scopus (1778) Google Scholar). The two enzymes have been designated isoenzymes βI and βII, and have isoelectric points of 8.9 and 9.0, respectively. Amino acid sequence analyses reveal a single amino acid difference in the first 50 NH2-terminal amino acid residues, and the complete amino acid sequence of isoenzyme βII has been deduced from the nucleotide sequence of a corresponding cDNA clone (2Leah R. Kigel J. Svendsen I. Mundy J. J. Biol. Chem. 1995; 270: 15789-15797Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). The function of the β-glucosidases in the germinated barley grain has not been defined unequivocally, but will clearly be related to their substrate specificities. It has been widely assumed that β-glucosidases prefer substrates of the type G-O-X, where G indicates the glucosyl residue and X can either be another glycosyl residue, for which the linkage position is not crucial, or a non-glycosyl aglycone group. As a result of the capacity of many β-glucosidases to hydrolyze glucosides with a range of glycosyl or non-glycosyl aglycone groups, non-physiological substrates such as 4-nitrophenyl β-d-glucopyranoside (4-NPG) 1The abbreviations used are: 4-NPG, 4-nitrophenyl β-d-glucopyranoside; BSA, bovine serum albumin; DP, degree of polymerization; EAC, 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide; GEE, glycine ethyl ester; G4G3Gred (where G indicates a glucosyl residue, the numbers represent linkage types, and red is the reducing end), 3-O-β-cellobiosyl-d-glucose; G4G4G3Gred, 3-O-β-cellotriosyl-d-glucose; HCA, hydrophobic cluster analysis; 4-NP, 4-nitrophenyl; β-glucosidase, β-d-glucoside glucohydrolase (EC 3.2.1.21); PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography. have been synthesized to measure activity in convenient spectrophotometric assays; the barley enzymes have also been assayed in this way. It has further been assumed that the rate of hydrolysis of oligomeric substrates by β-glucosidases will remain approximately constant or decrease with increasing degree of polymerization (DP) of the substrate (5Reese E.T. Maguire A.H. Parrish F.W. Can. J. Biochem. 1968; 46: 25-34Crossref PubMed Scopus (102) Google Scholar). However, the barley β-glucosidase isoenzyme βII hydrolyzes (1,4)-β-oligoglucosides much more efficiently than it hydrolyzes the aryl-β-glucoside 4-NPG (3Hrmova M. Harvey A.J. Wang J. Shirley N.J. Jones G.P. Stone B.A. Høj P.B. Fincher G.B. J. Biol. Chem. 1996; 271: 5277-5286Abstract Full Text PDF PubMed Google Scholar). The increased hydrolytic rate with oligosaccharides is a characteristic often observed with polysaccharide exohydrolases (5Reese E.T. Maguire A.H. Parrish F.W. Can. J. Biochem. 1968; 46: 25-34Crossref PubMed Scopus (102) Google Scholar), although the barley β-glucosidase is not able to hydrolyze polymeric (1,4)-β-glucans (2Leah R. Kigel J. Svendsen I. Mundy J. J. Biol. Chem. 1995; 270: 15789-15797Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 3Hrmova M. Harvey A.J. Wang J. Shirley N.J. Jones G.P. Stone B.A. Høj P.B. Fincher G.B. J. Biol. Chem. 1996; 271: 5277-5286Abstract Full Text PDF PubMed Google Scholar). These apparent anomalies led Hrmova et al. (3Hrmova M. Harvey A.J. Wang J. Shirley N.J. Jones G.P. Stone B.A. Høj P.B. Fincher G.B. J. Biol. Chem. 1996; 271: 5277-5286Abstract Full Text PDF PubMed Google Scholar) to suggest that the barley enzyme could be classified either as a polysaccharide exohydrolase of the (1,4)-β-glucan glucohydrolase group (EC 3.2.1.74) or as a β-glucosidase of the EC 3.2.1.21 class. Because β-glucosidases are widely distributed in nature, a precise understanding of substrate specificity and the mechanisms of substrate binding and catalysis is essential for defining the functions and tracing the evolution of this important group of enzymes. Here, subsite binding energies of barley β-glucosidase isoenzyme βII have been calculated from kinetic data during the hydrolysis of a series of β-oligoglucoside substrates. The analyses indicate that the enzyme has at least six glucosyl-binding subsites. The catalytic amino acids have been defined, together with their disposition in the substrate-binding region. The substrate specificity, action pattern, putative catalytic residues, and subsite mapping data can all be reconciled with a three-dimensional model of the barley β-glucosidase, which is based on the x-ray crystal structure of an homologous cyanogenic β-glucosidase from white clover (6Barrett T. Suresh C.G. Tolley S.P. Dodson E.J. Hughes M.A. Structure. 1995; 3: 951-960Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). The glucose diagnostic kit, 4-NPG, gentiobiose, linamarin, bovine serum albumin (BSA), dithiothreitol, glycine ethyl ester (GEE), and orcinol were purchased from Sigma. Conduritol B epoxide was from ICN (Costa Mesa, CA), [14C]GEE was from American Radiolabeled Chemicals (St. Louis, MO),l-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin was from Worthington, trifluoroacetic acid was from Pierce, and Microcon microconcentrators were from Amicon (Beverly, MA). The Brownlee C18 guard column was obtained from Applied Biosystems (Foster City, CA), a W-Porex C18 analytical column was from Phenomenex (Torrance, CA), Kieselgel 60 thin layer plates and sodium 2,2-dimethyl-2-silapentane-5-sulfonic acid were from Merck (Darmstadt, Germany), chromatography paper no. 3MM Chr was from Whatman (Maidstone, Kent, United Kingdom), and (1,4)-β-oligoglucosides (cellodextrins) of DP 2–6 and (1,3)-β-oligoglucosides (laminaridextrins) of DP 2–7 were from Seikagaku Kogyo Co. (Tokyo, Japan). Barley β-glucosidase isoenzyme βII was purified from a homogenate of 8-day-old seedlings as described previously (3Hrmova M. Harvey A.J. Wang J. Shirley N.J. Jones G.P. Stone B.A. Høj P.B. Fincher G.B. J. Biol. Chem. 1996; 271: 5277-5286Abstract Full Text PDF PubMed Google Scholar). Its purity was assessed by SDS-PAGE, where a single protein band was detected at high protein loadings. The purity was further confirmed by NH2-terminal amino acid sequence analysis; no secondary sequences were detected, and recoveries were very close to theoretical values (data not shown). Protein determination during the purification process, SDS-PAGE, and amino acid sequence analyses were performed as described previously (3Hrmova M. Harvey A.J. Wang J. Shirley N.J. Jones G.P. Stone B.A. Høj P.B. Fincher G.B. J. Biol. Chem. 1996; 271: 5277-5286Abstract Full Text PDF PubMed Google Scholar). Kinetic parameters on (1,4)-β- and (1,3)-β-oligoglucosides were measured at 37 °C by incubating 2–7 pmol of the purified β-glucosidase in 100 mm sodium acetate buffer, pH 5, containing 160 μg/ml BSA. Initial rates of hydrolysis were determined in triplicate at substrate concentrations ranging from 0.2 to 4 times the K m value. Enzymic reactions were stopped by heating to 100 °C for 2 min, and released glucose was measured by the glucose oxidase method (7Raabo E. Takildson T.C. Scand. J. Clin. Lab. Invest. 1960; 12: 402-407Crossref PubMed Scopus (597) Google Scholar). Standard deviations, which ranged between 1% and 7%, were calculated (8Samuels M.L. Statistics for the Life Sciences. Maxwell Macmillan International Editions/Dallen Publishing Co., San Francisco1991Google Scholar), and kinetic data were processed by a proportional weighted fit, using a nonlinear regression analysis program based on the Michaelis-Menten model equation (9Perella F. Anal. Biochem. 1988; 174: 437-447Crossref PubMed Scopus (280) Google Scholar). The initial enzyme concentration [E]0 was kept very much lower than the initial substrate concentration [S]0, and care was taken to measure initial reactions rates in all cases (10Hrmova M. Garrett T.P.J. Fincher G.B J. Biol. Chem. 1995; 270: 14556-14563Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Initial hydrolysis rates of β-glucosidase isoenzyme βII with the synthesized aryl-glycosides 4-NP-β-laminaribioside, 4-NP-β-cellobioside, and 4-NP-β-gentiobioside, and with 4-NPG as substrates, were measured at 1 mm concentration. Substrate solutions were prepared in 50 mm sodium acetate buffer, pH 5.0, using Δε (300 nm) of 1.104m−1 cm−1 to measure concentrations, and were incubated with 4–5 pmol of the purified β-glucosidase at 37 °C. Enzyme activity was determined reductometrically by monitoring the increase in reducing sugars or spectrophotometrically at 410 nm with 4-NPG (3Hrmova M. Harvey A.J. Wang J. Shirley N.J. Jones G.P. Stone B.A. Høj P.B. Fincher G.B. J. Biol. Chem. 1996; 271: 5277-5286Abstract Full Text PDF PubMed Google Scholar). One unit of activity is defined as the amount of enzyme required to release 1 μmol of glucose from aryl-glycosides or to release 1 μmol of 4-nitrophenol from 4-NPG per min. One unit corresponds to 16.67 nanokatals. Activity on linamarin was measured as described by Barrett et al.(6Barrett T. Suresh C.G. Tolley S.P. Dodson E.J. Hughes M.A. Structure. 1995; 3: 951-960Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Subsite affinities of β-glucosidase were calculated from Michaelis constants (K m ) and catalytic rate constants (k cat) during the hydrolysis of (1,4)-β-oligoglucosides of DP 2–6. In addition, the affinities of subsites +2 and +3 for (1,3)-β-linked oligoglucosides were calculated using K m and k cat values obtained with (1,3)-β-oligoglucosides of DP 2–4. The calculation procedures, using equations for the subsite analysis of exo-acting enzymes (11Hiromi K. Nitta Y. Numata C. Ono S. Biochem. Biophys. Acta. 1973; 302: 362-375Crossref PubMed Scopus (233) Google Scholar) but using subsite designations from −1 to +1, +2, etc., are summarized below. Consider an active site consisting of x subsites, labeled −1, +1, … to (x − 1). The subsite affinityA i of the subsite labeled i (1 <i ≤ (x − 1)) can be calculated from Equation 1. An=∑i=−1nAi−∑i=−1n−1Ai=RT[ln(kcat/Km)n+1−ln(kcat/Km)n]Equation 1 ∑i =−1n(Ai) and ∑i =−1n −1(Ai) represent the sums of subsite affinities for subsites labeled from −1 to n and n − 1, respectively. The parameters (k cat/K m ) n +1and (k cat/K m ) n are determined for oligosaccharides of DP n + 1 andn, respectively; R is the gas constant, andT is the absolute temperature. The valuesA −1 and k int are extrapolated from a plot of exp( An/RT ) versus(1/k cat) n . exp(An/RT)=[kint/(kcat)n−1]·exp(A−1/RT)Equation 2 The vertical and horizontal intercepts of this equation are −exp( A −1/ RT ) and 1/k int. The parameterk int is the intrinsic catalytic rate constant, which is independent of DP. Finally, A +1 is calculated from Equation 3. A+1=RT ln[(kcat/Km)n/0.018·kint)]−[A−1+A+2+…+An−1]Equation 3 The validity of the subsite map was subsequently confirmed by comparing the experimental and theoreticalk cat/K m values. The theoretical values were calculated according to Equation 4. (kcat/Km)n=(0.018)kint·exp∑−1n−1Ai/RTEquation 4 The equation uses the A i andk int parameters obtained for the (1,4)-β-oligoglucosides (Table I). The comparison of the experimental and theoretical k cat/K m values gave a maximum deviation of 0.4%, which confirms the validity of the model and the calculations of subsite affinities.Table IKinetic parameters K m, k cat, and k cat /K m of β-glucosidase isoenzyme βII during the hydrolysis of cellooligosaccharides of DP 2–6 and laminarioligosaccharides of DP 2–4DPK mk catk cat/K mmms −110 −3 s −1 m −1Cellooligosaccharides 22.67 ± 0.1911.58 ± 0.634.34 ± 0.07 30.97 ± 0.061.95 ± 0.122.01 ± 0.01 40.89 ± 0.058.88 ± 0.589.98 ± 0.08 50.41 ± 0.0211.66 ± 0.7628.44 ± 0.44 60.29 ± 0.0211.80 ± 0.7740.69 ± 0.14Laminarioligosaccharides 25.37 ± 0.3814.14 ± 1.022.63 ± 0.01 32.77 ± 0.201.44 ± 0.100.52 ± 0.01 40.52 ± 0.040.01 ± 0.0010.02 ± 0.001 Open table in a new tab Freshly prepared 4-NPG (100 mm) in 20 mm sodium acetate buffer, pH 5.0, was incubated with 2–3 pmol of the purified β-glucosidase for up to 72 h at 37 °C. The reaction was stopped by heating to 100 °C for 2 min. Aliquots of the reaction mixture were separated by thin layer chromatography on Kieselgel 60 thin layer plates and developed in ethyl acetate/acetic acid/H2O (5:2:1, by volume). Reducing sugars were detected using the orcinol reagent (12Hrmova M. Fincher G.B. Biochem. J. 1993; 289: 453-461Crossref PubMed Scopus (103) Google Scholar). Individual products were scraped off the plates, eluted from the Kieselgel with water, and their relative abundance determined spectrophotometrically at 300 nm using Δε of 1.104m−1 cm−1. For structural analysis of the transglycosylation products, the reaction mixtures were scaled up 5 times and products were separated by descending paper chromatography on Whatman no. 3MM paper in ethyl acetate/acetic acid/H2O (3:2:1, by volume) at ambient temperature. The products were excised from the chromatogram, eluted from the paper with water, concentrated under reduced pressure, and analyzed using a Perkin-Elmer Sciex PAI 300 electrospray ionization triple quadrupole mass spectrometer (Perkin Elmer Sciex Instruments, Thornhill, Ontario, Canada) and by 13C NMR spectroscopy. 13C NMR spectra of oligosaccharides (3–8 μmol) were measured on a Varian Gemini 300 multinuclear spectrometer using 5-mm external diameter sample tubes at a probe temperature of 297 K. Transients (32Rixon J.E. Ferreira L.M.A. Durrant A.J. Laurie J.I. Hazlewood G.P. Gilbert H.J. Biochem. J. 1992; 285: 947-955Crossref PubMed Scopus (42) Google Scholar) were collected into 16,000 data points using a spectral width of 4.5 KHz and a relaxation delay of 3 s with a 45° pulse width. No Gaussian weight factor or line broadening was applied to the data before Fourier transformation. Spectra were referenced and chemical shifts (ppm) were given using sodium 2,2-dimethyl-2-silapentane-5-sulfonic acid as an external standard. Inactivation of β-glucosidase isoenzyme βII was monitored at 37 °C by incubating 57 pmol of purified enzyme in 100 mm sodium acetate buffer, pH 5.0, containing 160 μg/ml BSA, with 0–10 mm conduritol B epoxide. To stop the inactivation and to determine the residual activity at different times, 5-μl aliquots of the reaction mixture were diluted into 250 μl of 0.2% (w/v) 4-NPG in 100 mm sodium acetate buffer, pH 5.0, containing 160 μg/ml BSA. The residual enzyme activity was monitored spectrophotometrically at 410 nm. First-order rate constants (k app) were determined from the semi-logarithmic plots of residual activity as a function of time, using Equation5. ln(At/A0)=−kapp·tEquation 5 A t and A 0 are enzyme activities at time t and 0, respectively. TheK i and k max constants were determined and the order of the inactivation reaction was estimated (13Levy H.M. Leber P.D. Ryan E.M. J. Biol. Chem. 1963; 238: 3654-3659Abstract Full Text PDF PubMed Google Scholar) to yield a inhibitor:enzyme stoichiometry of 0.7 (± 0.1) (data not shown). β-Glucosidase isoenzyme βII (43 μg) was inactivated at 37 °C by incubating the purified enzyme in 100 mm sodium acetate buffer, pH 5.0, in the presence of 24 mm conduritol B epoxide; this corresponded to 4.4 times the K i value. The inactivated enzyme, which lost 99% of its activity over 5 h, was concentrated, and excess inhibitor was removed in a microconcentrator (exclusion limit 10,000). Native and conduritol B epoxide-inactivated enzymes were denatured and digested with trypsin, and the resulting tryptic peptides purified by HPLC and sequenced essentially as described by Chen et al.(14Chen L. Fincher G.B. Høj P.B. J. Biol. Chem. 1993; 268: 13318-13326Abstract Full Text PDF PubMed Google Scholar). The tryptic peptide-inhibitor conjugate (20 pmol), which was unique to the conduritol B epoxide-inactivated enzyme, was treated with 20 μl of ammonium hydroxide at 50 °C for 20 min to remove covalently bound conduritol. Excess ammonia was removed under vacuum, and the peptide was again subjected to NH2-terminal amino acid sequence analysis. Purified enzyme (57 pmol) was inactivated with 15 mm1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide (EAC) in the presence of 125 mm [14C]GEE, the inactivated enzyme was denatured and digested with trypsin, and the resultant tryptic peptides were purified by HPLC for amino acid sequence analysis (14Chen L. Fincher G.B. Høj P.B. J. Biol. Chem. 1993; 268: 13318-13326Abstract Full Text PDF PubMed Google Scholar). In addition, the molecular mass of the HPLC-purified peptide-inhibitor conjugate was estimated on a Finnigan Lasermat 2000 matrix-assisted laser desorption ionization time-of-flight mass spectrometer (Finnigan MAT, Hemel Hempstead, UK). Hydrophobic cluster analysis (HCA) plots were obtained using standard computer software and interpreted as described by Gaboriaud et al. (15Gaboriaud C. Bissery V. Benchetrit T. Mornon J.P. FEBS Lett. 1987; 224: 149-155Crossref PubMed Scopus (542) Google Scholar). The amino acid sequence of the barley β-glucosidase isoenzyme βII was taken from Leah et al. (2Leah R. Kigel J. Svendsen I. Mundy J. J. Biol. Chem. 1995; 270: 15789-15797Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar), and the sequence of the cyanogenic β-glucosidase from white clover (Trifolium repens) was obtained from the Brookhaven Protein Data Bank (entry 1cbg). Co-ordinates for a three-dimensional model of the barley β-glucosidase were obtained using Swiss-Model, the automated protein modeling service of the ExPasy Molecular Biology Server (16Peitsch M.C. Jongeneel C.V. Int. Immunol. 1993; 5: 233-238Crossref PubMed Scopus (153) Google Scholar, 17Peitsch M.C. Bio/Technology. 1995; 13: 658-660Crossref Scopus (116) Google Scholar, 18Peitsch M.C. Biochem. Soc. Trans. 1996; 24: 274-279Crossref PubMed Scopus (900) Google Scholar). The barley enzyme sequence and co-ordinates of the white clover β-glucosidase (6Barrett T. Suresh C.G. Tolley S.P. Dodson E.J. Hughes M.A. Structure. 1995; 3: 951-960Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar) (Brookhaven Data Bank entry 1cbg) were supplied as inputs for the program. The three-dimensional model of cellooctaose was constructed from the co-ordinates of cellobiose (19Raymond S. Heyraud A. Tran-Qui D. Krick A. Chanzy H. Macromolecules. 1995; 28 (2010): 2096Crossref Scopus (64) Google Scholar). Fitting the cellooctaose model into the active site pocket of the barley β-glucosidase model was performed on a Silicon Graphics Iris Indigo Elan 4000 work station using the GRASP (20Nicholls A. Sharp K.A. Honig B. Proteins Struct. Funct. Genet. 1991; 4: 281-296Crossref Scopus (5318) Google Scholar) and O (21Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A Crystallogr. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar) software programs. Co-ordinates of the Clostridium thermocellumendoglucanase CelC were obtained from the Brookhaven Protein Data Bank (entry 1cen; CelCE140Q-Glc6). Protein models and stereoview diagrams were generated with the RasMol software program (22Sayle R. RasMol Version 2.6 Molecular Visualization Program. Glaxo Wellcome Research Development, Stevenage, United Kingdom1996Google Scholar). The relationship between the kinetic parameters (K m, k cat, andk cat/K m ) and the DPs of oligosaccharide substrates of the barley β-glucosidase isoenzyme βII are shown in Table I. For cellodextrin substrates, K m decreases with increasing chain length of the substrate, whilek cat values appear to be relatively independent of DP, except in the case of cellotriose and to a lesser extent cellotetraose, where k cat values are somewhat lower (Table I). Catalytic efficiency factorsk cat/K m increase steadily with increasing DP of the (1,4)-β-oligoglucoside substrates, again with the exception of cellotriose. Indeed, thek cat/K m value for cellohexaose is 9-fold higher than the value for cellobiose. In marked contrast to the hydrolysis of the cellodextrin series, thek cat andk cat/K m values for laminaridextrins decreased by over 1000-fold and over 130-fold, respectively, as the DP increased from 2 to 4 (Table I). The rate of hydrolysis of laminaripentaose and longer (1,3)-β-oligoglucosides was too low to allow the precise measurement of kinetic parameters. The enzyme had no activity on the cyanogenic substrate, linamarin. Binding energies for the six β-glucosyl binding subsites in the barley β-glucosidase isoenzyme βII during hydrolysis of cellodextrins are compared in Fig. 1. Hrmova et al. (3Hrmova M. Harvey A.J. Wang J. Shirley N.J. Jones G.P. Stone B.A. Høj P.B. Fincher G.B. J. Biol. Chem. 1996; 271: 5277-5286Abstract Full Text PDF PubMed Google Scholar) reported that the enzyme catalyzes the hydrolytic removal of glucose units from the non-reducing termini of oligosaccharide chains; this demonstrated that the enzyme is an exohydrolase. It is clear, therefore, that the catalytic amino acids are located between the non-reducing terminal glucosyl-binding subsite and the penultimate subsite; these are designated subsites −1 and +1, respectively (Fig. 1). Binding energies have been expressed in the past as positive or negative values (23Allen J.D. Thoma J.A. Biochem. J. 1976; 159: 121-132Crossref PubMed Scopus (58) Google Scholar, 24Suganuma T. Matsuno R. Ohnishi M. Hiromi K. J. Biochem. (Tokyo). 1978; 84: 293-316Crossref PubMed Scopus (136) Google Scholar, 25Biely P. Vrsanska M. Claeyssens M. Eur. J. Biochem. 1991; 200: 157-163Crossref PubMed Scopus (100) Google Scholar), but we prefer to use positive values to indicate binding (10Hrmova M. Garrett T.P.J. Fincher G.B J. Biol. Chem. 1995; 270: 14556-14563Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) (Fig. 1). The negative value observed at subsite +2 indicates that there is a degree of repulsion between the enzyme and the glucosyl residue at this subsite. The binding energies at subsites +3, +4, and +5 are positive, but decrease in magnitude as the distance from the catalytic site increases (Fig. 1). Although the barley β-glucosidase hydrolyzes laminaribiose at a slightly faster rate than cellobiose, the rate decreases steeply as the DP of the (1,3)-β-oligoglucoside substrates increase (Table I). Calculation of the apparent binding energies of these laminaridextrins to subsites +2 and +3 yielded values of −4.2 and −8.4 kJ·mol−1, consistent with the low affinities andk cat values of the enzyme for laminaritriose and laminaritetraose (Table I). The subsite mapping results (Fig. 1) offer an explanation for the relatively low rate of hydrolysis of cellotriose (Table I). Based on the binding energies of individual subsites, one might anticipate that “non-productive” binding of cellotriose across subsites +1 to +3 would be almost as likely to occur on thermodynamic grounds as would “productive” binding from subsites −1 to +2. Because the non-productive binding does not span the catalytic site, hydrolysis will not occur, but occupation of part of the substrate-binding region would be expected to lower the catalytic rate, and hence the catalytic efficiency (Table I). Non-productive binding of cellotetraose in subsites +1 to +4 might also occur, but to a lesser extent. When the barley β-glucosidase was incubated with 100 mm 4-NPG, the major products were glucose and 4-NP, as expected. However, significant amounts of other, higher molecular weight 4-NP derivatives were visible on thin layer chromatograms (data not shown). When the three most abundant of these were eluted from the plates and analyzed, m/z values of 486.2 or 486.3 were obtained by electrospray mass spectrometry (data not shown). These values correspond to disaccharide derivatives of 4-NP. Chemical shifts obtained for 13C NMR spectroscopy indicated that the disaccharide derivatives were 4-NP-β-laminaribioside, 4-NP-β-cellobioside, and 4-NP-β-gentiobioside (Table II).Table II13 C NMR chemical shifts of aryl-glycosides synthesized by β-glucosidase isoenzyme βII during the glycosyl transfer reaction of 100 mm 4-NPGCompoundCarbonNon-reducing residueResidue carrying the aglycon4-NP-β-laminaribioside1104.1100.5274.773.8376.885.1470.869.1577.377.2662.061.84-NP-β-cellobioside1103.8100.5273.874.4376.475.3470.779.4577.376.7661.961.04-NP-β-gentiobioside1104.0100.5273.874.3376.776.5470.970.4577.176.9661.969.6 Open table in a new tab The relative proportions of individual transglycosylation products are compared in Table III. The 4-NP-β-laminaribioside was the most abundant, but significant levels of 4-NP-β-cellobioside and 4-NP-β-gentiobioside were also detected. This suggests that there is some flexibility in the binding of a glucosyl residue to subsite +1, because the 4-NPG is able to move sufficiently in the active site to present hydroxyls on C atoms 3, 4, or 6 to the bound glycone prior to transfer of that bound glucosyl residue to the 4-NPG (Scheme FSI). The purified transglycosylation products were subsequently used as substrates under standard conditions for hydrolysis. The specific activities of hydrolysis were found to reflect, approximately at least, their relative rates of synthesis under conditions that promote glycosyl transfer reactions (Table III, column 3; cf. column 2).Table IIISummary of properties of glycosyl-transfer and other reaction products produced by β-glucosidase isoenzyme βII during the glycosyl transfer reaction of 100 mm 4-NPGSubstrate or reaction productChromatographic mobility3-aMobilities of aryl-glycosides and other carbohydrates relative to Glc mobility.Distribution after 72 h3-bDistribution of aryl-glycosides and other reaction products in percent of initial substrate concentration.Specific hydrolytic activity3-cSpecific hydrolytic activity of the β-glucosidase at 1 mm concentration of aryl-glycosides.R Glc%units/mg4-NPG2.2240.64-NP-β-laminaribioside1.8102.04-NP-β-cellobioside1.661.24-NP-β-gentiobioside1.430.3Other arylglycosides1.1–1.24NA3-1004Not applicable.Glc148NA3-1004Not applicable.Disaccharides0.4–0.65NA3-1004Not applicable.3-a Mobilities of aryl-glycosides and other carbohydrates relative to Glc mobility.3-b Distribution of aryl-glycosides and other reaction products in percent of initial substrate concentration.3-c Specific hydrolytic activity of the β-glucosidase at 1 mm concentration of aryl-glycosides.3-d Not applicable. Open table in a new tab Conduritol B epoxide, or 1,2-anhydro-myo-inositol, has been" @default.
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• W2012194120 title "Substrate Binding and Catalytic Mechanism of a Barley β-d-Glucosidase/(1,4)-β-d-Glucan Exohydrolase" @default.
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