Enzymatic characteristics of cellobiose phosphorylase from Ruminococcus albus NE1 and kinetic mechanism of unusual substrate inhibition in reverse phosphorolysis

Cellobiose phosphorylase (CBP) catalyzes the reversible phosphorolysis of cellobiose to produce α-D-glucopyranosyl phosphate (Glc1P) and D-glucose. It is an essential enzyme for the metabolism of cello-oligosaccharides in a ruminal bacterium, Ruminococcus albus. In this study, recombinant R. albus CBP (RaCBP) produced in Escherichia coli was characterized. It showed highest activity at pH 6.2 at 50 °C, and was stable in a pH range of 5.5-8.8 and at below 40 °C. It phosphorolyzed only cellobiose efficiently, and the reaction proceeded through a random-ordered bi bi mechanism, by which inorganic phosphate and cellobiose bind in random order and D-glucose is released before Glc1P. In the synthetic reaction, RaCBP showed highest activity to D-glucose, followed by 6-deoxy-D-glucose. D-Mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, 1,5-anhydro-D-glucitol, and gentiobiose also served as acceptors, although the activities for them were much lower than for D-glucose. D-Glucose acted as a competitive-uncompetitive inhibitor of the reverse synthetic reaction, which bound not only the Glc1P site (competitive) but also the ternary enzyme-Glc1P-D-glucose complex (uncompetitive).     

Characterization of a cellobiose phosphorylase from a hyperthermophilic eubacterium,Thermotoga maritima MSB8

The cepA putative gene encoding a cellobiose phosphorylase of Thermotoga maritima MSB8 was cloned, expressed in Escherichia coli BL21-codonplus-RIL and characterized in detail. The maximal enzyme activity was observed at pH 6.2 and 80 degrees C. The energy of activation was 74 kJ/mol. The enzyme was stable for 30 min at 70 degrees C in the pH range of 6-8. The enzyme phosphorolyzed cellobiose in an random-ordered bi bi mechanism with the random binding of cellobiose and phosphate followed by the ordered release of D-glucose and alpha-D-glucose-1-phosphate. The Km for cellobiose and phosphate were 0.29 and 0.15 mM respectively, and the kcat was 5.4 s(-1). In the synthetic reaction, D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, and 6-deoxy-D-glucose were found to act as glucosyl acceptors. Methyl-beta-D-glucoside also acted as a substrate for the enzyme and is reported here for the first time as a substrate for cellobiose phosphorylases. D-Xylose had the highest (40 s(-1)) kcat followed by 6-deoxy-D-glucose (17 s(-1)) and 2-deoxy-D-glucose (16 s(-1)). The natural substrate, D-glucose with the kcat of 8.0 s(-1) had the highest (1.1 x 10(4) M(-1) s(-1)) kcat/Km compared with other glucosyl acceptors. D-Glucose, a substrate of cellobiose phosphorylase, acted as a competitive inhibitor of the other substrate, alpha-D-glucose-1-phosphate, at higher concentrations.     

Synthetic reaction of Cellvibrio gilvus cellobiose phosphorylase.

The synthetic reactions of the cellobiose phosphorylase from Cellvibrio gilvus were investigated in detail. It was found that, besides D-glucose, some sugars having substitution or deletion of the hydroxyl group at C2 or C6 of the D-glucose molecule could serve as a glucosyl acceptor, though less effectively than D-glucose. The enzyme showed higher activity with beta-D-glucose than with the alpha-anomer as an acceptor. This result indicates that it recognizes the anomeric hydroxyl group not involved directly in the reaction. beta-D-Cellobiose was also phosphorolyzed faster than the alpha-anomer. Substrate inhibition was observed with D-glucose, 6-deoxy-D-glucose, or D-glucosamine as an acceptor, with D-glucose being most inhibiting. This inhibition was studied in detail and it was found that D-glucose competes with alpha-D-glucose-1-phosphate for its binding site. A model of competitive substrate inhibition was proposed, and the experimental data fit well to the theoretical values that were calculated in accordance with this model.     

Structural dissection of the reaction mechanism of cellobiose phosphorylase

Cellobiose phosphorylase, a member of the glycoside hydrolase family 94, catalyses the reversible phosphorolysis of cellobiose into alpha-D-glucose 1-phosphate and D-glucose with inversion of the anomeric configuration. The substrate specificity and reaction mechanism of cellobiose phosphorylase from Cellvibrio gilvus have been investigated in detail. We have determined the crystal structure of the glucose-sulphate and glucose-phosphate complexes of this enzyme at a maximal resolution of 2.0 A (1 A=0.1 nm). The phosphate ion is strongly held through several hydrogen bonds, and the configuration appears to be suitable for direct nucleophilic attack to an anomeric centre. Structural features around the sugar-donor and sugar-acceptor sites were consistent with the results of extensive kinetic studies. When we compared this structure with that of homologous chitobiose phosphorylase, we identified key residues for substrate discrimination between glucose and N-acetylglucosamine in both the sugar-donor and sugar-acceptor sites. We found that the active site pocket of cellobiose phosphorylase was covered by an additional loop, indicating that some conformational change is required upon substrate binding. Information on the three-dimensional structure of cellobiose phosphorylase will facilitate engineering of this enzyme, the application of which to practical oligosaccharide synthesis has already been established.     

Enzymic Preparation of Benzyl β-D-Glucopyranoside

Benzyl β-D-glucopyranoside was prepared by an enzyme-catalysed direct reaction between D-glucose, or better cellobiose, and benzyl alcohol in the presence of a minimum amount of water. The enzyme β-glucosidase was used in the immobilized form (adsorbed onto macroporous polyethylene terephthalate or covalently bound on polyglycidyl methacrylate), enabling multiple application.     

Computational analyses of the conformational itinerary along the reaction pathway of GH94 cellobiose phosphorylase

GH94 cellobiose phosphorylase (CBP) catalyzes the phosphorolysis of cellobiose into α-d-glucose 1-phosphate (G1P) and d-glucose with inversion of anomeric configuration. The complex crystal structure of CBP from Cellvibrio gilvus had previously been determined; glycerol, glucose, and phosphate are bound to subsites −1, +1, and the anion binding site, respectively. We performed computational analyses to elucidate the conformational itinerary along the reaction pathway of this enzyme. autodock was used to dock cellobiose with its glycon glucosyl residue in various conformations and with its aglycon glucosyl residue in the low-energy ⁴C₁ conformer. An oxocarbenium ion-like glucose molecule mimicking the transition state was also docked. Based on the clustering analysis, docked energies, and comparison with the crystallographic ligands, we conclude that the reaction proceeds from ¹S₃ as the pre-transition state conformer (Michaelis complex) via E₃ as the transition state candidate to ⁴C₁ as the G1P product conformer. The predicted reaction pathway of the inverting phosphorylase is similar to that proposed for the first-half glycosylation reaction of retaining cellulases, but is different from those for inverting cellulases. NAMD was used to simulate molecular dynamics of the enzyme. The ¹S₃ pre-transition state conformer is highly stable compared with other conformers, and a conformational change from ⁴C₁ to ^1,4B was observed.     

Purification and characterization of NADP+-dependent 3 alpha-hydroxysteroid dehydrogenase from mouse liver cytosol

A monomeric 3 alpha-hydroxysteroid dehydrogenase with a molecular weight of 34,000 was purified to apparent homogeneity from mouse liver cytosol. The enzyme catalyzed the reversible oxidation of the 3 alpha-hydroxy group of C19-, C21-, and C24-steroids, reduced a variety of carbonyl compounds, and was inhibited by SH-reagents, synthetic estrogens, anti-inflammatory drugs, prostaglandins, and delta 4-3-ketosteroids. Although these properties are similar to those of the enzyme from rat liver cytosol, the mouse enzyme exhibited low dehydrogenase activity toward benzene dihydrodiol and some alicyclic alcohols, it showed a strict cofactor specificity for NADP(H), and high substrate inhibition was observed in the reverse reaction. In addition, dexamethasone, deoxycorticosterone, and medroxyprogesterone acetate inhibited the mouse enzyme competitively at low concentrations and noncompetitively at high concentrations, whereas hexestrol, indomethacin, and prostaglandin A1 were competitive inhibitors. Steady-state kinetic measurements in both directions indicated that the reaction proceeds through an ordered bi bi mechanism with the cofactors binding to the free enzyme. The 3-ketosteroid substrates inhibited the enzyme uncompetitively at elevated concentrations, suggesting that the substrates bind to the enzyme.NADPH complex and to the enzyme NADP+ complex.     

Characterization of a Cellobiose Phosphorylase from a Hyperthermophilic Eubacterium,Thermotoga maritima MSB8

The cepA putative gene encoding a cellobiose phosphorylase of Thermotoga maritima MSB8 was cloned, expressed in Escherichia coli BL21-codonplus-RIL and characterized in detail. The maximal enzyme activity was observed at pH 6.2 and 80°C. The energy of activation was 74 kJ/mol. The enzyme was stable for 30 min at 70°C in the pH range of 6-8. The enzyme phosphorolyzed cellobiose in an random-ordered bi bi mechanism with the random binding of cellobiose and phosphate followed by the ordered release of D-glucose and α-D-glucose-1-phosphate. The K _m for cellobiose and phosphate were 0.29 and 0.15 mM respectively, and the k _cat was 5.4 s^-1. In the synthetic reaction, D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, and 6-deoxy-D-glucose were found to act as glucosyl acceptors. Methyl-β-D-glucoside also acted as a substrate for the enzyme and is reported here for the first time as a substrate for cellobiose phosphorylases. D-Xylose had the highest (40 s^-1) k _cat followed by 6-deoxy-D-glucose (17 s^-1) and 2-deoxy-D-glucose (16 s^-1). The natural substrate, D-glucose with the k _cat of 8.0 s^-1 had the highest (1.1×10⁴ M^-1 s^-1) k _cat/K _m compared with other glucosyl acceptors. D-Glucose, a substrate of cellobiose phosphorylase, acted as a competitive inhibitor of the other substrate, α-D-glucose-1-phosphate, at higher concentrations.     

Beta-D-glucosidase reaction kinetics from isothermal titration microcalorimetry

The cellobiase activities of nine thermal stable mutants of Thermobifida fusca BglC were assayed by isothermal titration microcalorimetry (ITC). The mutations were previously generated using random mutagenesis and identified by high-temperature screening as imparting improved thermal stability to the beta-D-glucosidase enzyme. Analysis of the substrate-saturation curves obtained by ITC for the wild-type enzyme and the nine thermally stabilized mutants revealed that the wild type and all the mutants were subject to binding of a second substrate molecule. Furthermore, the "inhibited" enzyme-substrate complexes were shown to retain catalytic activity. In the case of three of the BglC mutants (N178I, N317Y/L444F, and N317Y/L444F/A433V), binding of a second substrate molecule resulted in improved cellobiose turnover rates at lower substrate concentrations. No correlation between denaturation temperatures of the mutants and activity on cellobiose at 25 degrees C was evident. However, one particular mutant, BglC S319C, was significantly improved in both thermal tolerance and cellobiase activity with respect to those of the wild-type BglC. The triple mutant, N317Y/L444F/A433V, had a 5 degrees C increase in denaturation temperature while maintaining activity levels similar to that of the wild type at higher substrate concentrations. ITC provided a highly sensitive and nondestructive means to continuously monitor the reaction of BglC with cellobiose, resulting in abundant data sets that could be rigorously analyzed by fitting to known enzyme kinetics models. One distinct advantage of using data from the ITC was the empirical validation of the pseudo steady state assumption, a necessary condition for obtaining solutions to the proposed mechanisms.     

Enzymic Preparation of Benzyl β-D-Glucopyranoside

Benzyl β-D-glucopyranoside was prepared by an enzyme-catalysed direct reaction between D-glucose, or better cellobiose, and benzyl alcohol in the presence of a minimum amount of water. The enzyme β-glucosidase was used in the immobilized form (adsorbed onto macroporous polyethylene terephthalate or covalently bound on polyglycidyl methacrylate), enabling multiple application.     

Kinetic mechanism of beta-glucosidase from Trichoderma reesei QM 9414

beta-Glucosidase is a key enzyme in the hydrolysis of cellulose to D-glucose. beta-Glucosidase was purified from cultures of Trichoderma reesei QM 9414 grown on wheat straw as carbon source. The enzyme hydrolyzed cellobiose and aryl beta-glucosides. The double-reciprocal plots of initial velocity vs. substrate concentration showed substrate inhibition with cellobiose and salicin. However, when p-nitrophenyl beta-D-glucopyranoside was the substrate no inhibition was observed. The corresponding kinetic parameters were: K = 1.09 +/- 0.2 mM and V = 2.09 +/- 0.52 mumol.min-1.mg-1 for salicin; K = 1.22 +/- 0.3 mM and V = 1.14 +/- 0.21 mumol.min-1.mg-1 for cellobiose; K = 0.19 +/- 0.02 mM and V = 29.67 +/- 3.25 mumol.min-1.mg-1 for p-nitrophenyl beta-D-glucopyranoside. Studies of inhibition by products and by alternative product supported an Ordered Uni Bi mechanism for the reaction catalyzed by beta-glucosidase on p-nitrophenyl beta-D-glucopyranoside as substrate. Alternative substrates as salicin and cellobiose, a substrate analog such as maltose and a product analog such as fructose were competitive inhibitors in the p-nitrophenyl beta-D-glucopyranoside hydrolysis.     

Anomeric and other substrate specificity studies with myo-inositol-1-P synthase

L-myo-Inositol-1-phosphate synthase has been found to have at least a 5-fold preference for the beta-anomer of its natural substrate D-Glc-6-P. The alpha-anomer appears to be an inhibitor of the reaction and may be converted to product as well. As well as showing an enzymatic preference for the equatorial C-1 hydroxyl of D-Glc-6-P, our results suggest that it is the pyranose form of D-Glc-6-P that binds to the enzyme and that ring-opening is an enzymatic step. We have also found D-2-dGlc-6-P, D-2-F-2-dGlc-6-P, and D-Man-6-P each to be both competitive inhibitors and substrates that are converted to inositol phosphates by the synthase. D-Allose-6-P is a weak inhibitor of the enzyme, but not a substrate. D-Gal-6-P is neither substrate nor inhibitor. Thus the specificity of the synthase with respect to single position epimers of D-Glc-6-P increases in the order C1 less than C2 much less than C3 less than C4.     

Cellotriose Synthesis Using d-Glucose as a Starting Material by Two Step Reactions of Immobilized β-Glucosidases

We tried to polymerize d-glucose to cellotriose, the smallest substrate for β-1,4-glucan synthesis by the β-transglycosylase of Trichoderma longibrachiatum, without participation of high energy compounds such as nucleotide sugars. A commercial β-glucosidase (sweet almond) showed a typical condensation reaction of d-glucose, producing cellobiose when it was entrapped in a visking tube and incubated in 30% d-glucose solution. The reaction was done with immobilized enzyme covalently bound to Polyacrylamide beads, and entrapped enzyme. Cellobiose (21.0 mg) was obtained from 30 g of d-glucose in a 3-day reaction, where 0.29 unit of the entrapped enzyme preparation was incubated with 100 ml of 30% d-glucose at pH 6.0 and 41°C. Gentiobiose was also produced in the mixture as a minor product. The immobilized β-glucosidase (Sumizyme C) preparation covalently bound to Polyacrylamide beads could catalyze a transglucosylation reaction to produce cellotriose from cellobiose in a good yield without production of gentiobiose. The transfer reaction was optimal at pH 4.8 and 30°C. Cellotriose (11.2 mg) was produced from the reaction mixture containing 68 mg of cellobiose and the enzyme preparation (0.1 unit) after 24-hr of incubation at the optimal conditions. Both immobilized β-glucosidases, sweet almond and Sumizyme C, may be used repeatedly without any loss of the initial activity.     

Crystal complex structures reveal how substrate is bound in the -4 to the +2 binding sites of Humicola grisea Cel12A

As part of an ongoing enzyme discovery program to investigate the properties and catalytic mechanism of glycoside hydrolase family 12 (GH 12) endoglucanases, a GH family that contains several cellulases that are of interest in industrial applications, we have solved four new crystal structures of wild-type Humicola grisea Cel12A in complexes formed by soaking with cellobiose, cellotetraose, cellopentaose, and a thio-linked cellotetraose derivative (G2SG2). These complex structures allow mapping of the non-covalent interactions between the enzyme and the glucosyl chain bound in subsites -4 to +2 of the enzyme, and shed light on the mechanism and function of GH 12 cellulases. The unhydrolysed cellopentaose and the G2SG2 cello-oligomers span the active site of the catalytically active H.grisea Cel12A enzyme, with the pyranoside bound in subsite -1 displaying a S31 skew boat conformation. After soaking in cellotetraose, the cello-oligomer that is found bound in site -4 to -1 contains a beta-1,3-linkage between the two cellobiose units in the oligomer, which is believed to have been formed by a transglycosylation reaction that has occurred during the ligand soak of the protein crystals. The close fit of this ligand and the binding sites occupied suggest a novel mixed beta-glucanase activity for this enzyme.     

Crystal structure of rabbit phosphoglucose isomerase complexed with its substrate D-fructose 6-phosphate.

Phosphoglucose isomerase (PGI, EC 5.3.1.9) catalyzes the interconversion of D-glucose 6-phosphate (G6P) and D-fructose 6-phosphate (F6P) and plays important roles in glycolysis and gluconeogenesis. Biochemical characterization of the enzyme has led to a proposed multistep catalytic mechanism. First, the enzyme catalyzes ring opening to yield the open chain form of the substrate. Then isomerization proceeds via proton transfer between C2 and C1 of a cis-enediol(ate) intermediate to yield the open chain form of the product. Catalysis proceeds in both the G6P to F6P and F6P to G6P directions, so both G6P and F6P are substrates. X-ray crystal structure analysis of rabbit and bacterial PGI has previously identified the location of the enzyme active site, and a recent crystal structure of rabbit PGI identified Glu357 as a candidate functional group for transferring the proton. However, it was not clear which active site amino acid residues catalyze the ring opening step. In this paper, we report the X-ray crystal structure of rabbit PGI complexed with the cyclic form of its substrate, D-fructose 6-phosphate, at 2.1 A resolution. The location of the substrate relative to the side chains of His388 suggest that His388 promotes ring opening by protonating the ring oxygen. Glu216 helps to position His388, and a water molecule that is held in position by Lys518 and Thr214 accepts a proton from the hydroxyl group at C2. Comparison to a structure of rabbit PGI with 5PAA bound indicates that ring opening is followed by loss of the protonated water molecule and conformational changes in the substrate and the protein so that a helix containing amino acids 513-520 moves in toward the substrate to form additional hydrogen bonds with the substrate.     

Preparation of cellulase concoction using differential adsorption phenomenon

Controlled depolymerization of cellulose is essential for the production of valuable cellooligosaccharides and cellobiose from lignocellulosic biomass. However, enzymatic cellulose hydrolysis involves multiple synergistically acting enzymes, making difficult to control the depolymerization process and generate desired product. This work exploits the varying adsorption properties of the cellulase components to the cellulosic substrate and aims to control the enzyme activity. Cellulase adsorption was favored on pretreated cellulosic biomass as compared to synthetic cellulose. Preferential adsorption of exocellulases was observed over endocellulase, while β-glucosidases remained unadsorbed. Adsorbed enzyme fraction with bound exocellulases when used for hydrolysis generated cellobiose predominantly, while the unadsorbed enzymes in the liquid fraction produced cellooligosaccharides majorly, owing to its high endocellulases activity. Thus, the differential adsorption phenomenon of the cellulase components can be used for the controlling cellulose hydrolysis for the production of an array of sugars.     

Structural basis for substrate binding and regioselective oxidation of monosaccharides at C3 by pyranose 2-oxidase

Pyranose 2-oxidase (P2Ox) participates in fungal lignin degradation by producing the H2O2 needed for lignin-degrading peroxidases. The enzyme oxidizes cellulose- and hemicellulose-derived aldopyranoses at C2 preferentially, but also on C3, to the corresponding ketoaldoses. To investigate the structural determinants of catalysis, covalent flavinylation, substrate binding, and regioselectivity, wild-type and mutant P2Ox enzymes were produced and characterized biochemically and structurally. Removal of the histidyl-FAD linkage resulted in a catalytically competent enzyme containing tightly, but noncovalently bound FAD. This mutant (H167A) is characterized by a 5-fold lower kcat, and a 35-mV lower redox potential, although no significant structural changes were seen in its crystal structure. In previous structures of P2Ox, the substrate loop (residues 452-457) covering the active site has been either disordered or in a conformation incompatible with carbohydrate binding. We present here the crystal structure of H167A in complex with a slow substrate, 2-fluoro-2-deoxy-D-glucose. Based on the details of 2-fluoro-2-deoxy-D-glucose binding in position for oxidation at C3, we also outline a probable binding mode for D-glucose positioned for regioselective oxidation at C2. The tentative determinant for discriminating between the two binding modes is the position of the O6 hydroxyl group, which in the C2-oxidation mode can make favorable interactions with Asp452 in the substrate loop and, possibly, a nearby arginine residue (Arg472). We also substantiate our hypothesis with steady-state kinetics data for the alanine replacements of Asp452 and Arg472 as well as the double alanine 452/472 mutant.     

Substrate-induced activation of maltose phosphorylase: interaction with the anomeric hydroxyl group of alpha-maltose and alpha-D-glucose controls the enzyme's glucosyltransferase activity

Maltose phosphorylase, long considered strictly specific for beta-D-glucopyranosyl phosphate (beta-D-glucose 1-P), was found to catalyze the reaction beta-D-glucosyl fluoride + alpha-D-glucose----alpha-maltose + HF, at a rapid rate, V = 11.2 +/- 1.2 mumol/(min.mg), and K = 13.1 +/- 4.4 mM with alpha-D-glucose saturating, at 0 degrees C. This reaction is analogous to the synthesis of maltose from beta-D-glucose 1-P + D-glucose (the reverse of maltose phosphorolysis). In acting upon beta-D-glucosyl fluoride, maltose phosphorylase was found to use alpha-D-glucose as a cosubstrate but not beta-D-glucose or other close analogs (e.g., alpha-D-glucosyl fluoride) lacking an axial 1-OH group. Similarly, the enzyme was shown to use alpha-maltose as a substrate but not beta-maltose or close analogs (e.g., alpha-maltosyl fluoride) lacking an axial 1-OH group. These results indicate that interaction of the axial 1-OH group of the disaccharide donor or sugar acceptor with a particular protein group near the reaction center is required for effective catalysis. This interaction appears to be the means that leads maltose phosphorylase to promote a narrowly defined set of glucosyl transfer reactions with little hydrolysis, in contrast to other glycosylases that catalyze both hydrolytic and nonhydrolytic reactions.     

Phosphoglycerate mutase. Kinetics and effects of salts on the mutase and bisphosphoglycerate phosphatase activities of the enzyme from chicken breast muscle.

The steady state kinetics and effects of salts on chicken breast phosphoglycerate mutase have been examined. The enzyme can catalyze three phosphoryl transfer reactions: mutase, bisphosphoglycerate phosphatase, and bisphosphoglycerate synthase. The mutase rate was measured in the favorable direction (Keq = glycerate-3-P/glycerate-2-P approximately equal to 12) using [2T]glycerate-2-P as substrate. The bisphosphoglycerate phosphatase activity was studied in the presence of the activator, glycolate-2-P. The latter is an analog of the glycerate-P's and appears to act as an abortive mutase substrate. The kinetic pattern obtained with both activities is that of a ping-pong mechanism with inhibition by the second substrate occurring at a lower concentration than the Km value for that substrate. The kinetic parameters for the mutase determined in 50 mM N-[tris(hydroxymethyl)methyl-2-amino]ethanesulfonate (TES)/sodium buffer containing 0.1 M KCl, pH 7.5, 25 degrees C are: Km glycerate-2,3-P2, 0.069 micron; Km glycerate-2-P, 14 micron; Km glycerate-3-P approximately 200 micron; Ki glycerate-2-P, 4 micron. The kinetic parameters for the phosphatase reaction in 50 mM triethanolamine/Cl- buffer, pH 7.5, 25 degrees C are: Km glycerate-2,3-P2, 0.065 micron:Km glycolate-2P, 479 micron; Ki glycolate-2-P, 135 micron. The enzyme is sensitive to changes in the ionic environment. Increasing salt concentrations activate the phosphatase in the presence of glycolate-2-P by decreasing the apparent Km of glycerate-2,3-P2. The effects are due to the anionic component and Cl- greater than acetate greater than TES. The same salts are competitive inhibitors with respect to glycolate-2-P. With high levels of KCl that produce a 30-fold decrease in the apparent maximal velocity due to competition with glycolate-2-P, the Km of glycerate-2,3-P2 remains low. These observations lead us to postulate that each monophosphoglycerate substrate has a separate site on the enzyme and that glycerate-2,3-P2 can bind to either site. The binding of anions to one site of the nonphosphorylated enzyme allows an increase in the on and off rates of glycerate-2,3-P2 at the alternate site. Salts inhibit the mutase reaction. The Km of glycerate-2,3-P2 is increased as is that of glycerate-2-P. The effect on the Km of glycerate-2,3-P2 is attributed to an increase in the off rate/on rate ratio for glycerate-2,3-P2. The bisphosphoglycerate synthase reaction is shown to require added glycerate-3-P. The equilibrium between enzyme and glycerate-1,3-P2 is favorable (Kdiss less than or equal 7 X 10(-8) M) and suggests that in the absence of a separate synthase this reaction may have functional significance.     

Crystal structures of Paenibacillus polymyxa beta-glucosidase B complexes reveal the molecular basis of substrate specificity and give new insights into the catalytic machinery of family I glycosidases

Bacteria species involved in degradation of cellulosic substrates produce a variety of enzymes for processing related compounds along the hydrolytic pathway. Paenibacillus polymyxa encodes two homologous beta-glucosidases, BglA and BglB, presenting different quaternary structures and substrate specificities. We previously reported the 3D-structure of BglA, which is highly specific against cellobiose. Here, we present structural analysis of BglB, a monomeric enzyme that acts as an exo-beta-glucosidase hydrolyzing cellobiose and cellodextrins of higher degree of polymerization. The crystal structure of BglB shows that several polar residues narrow the active site pocket and contour additional subsites. The structure of the BglB-cellotetraose complex confirms these subsites, revealing the substrate-binding mode, and shows the oligosaccharide-enzyme recognition pattern in detail. Comparison between BglA and BglB crystal structures suggests that oligomerization in BglA can assist in fine-tuning the specificity of the active centre by modulating the loops surrounding the cavity. We have solved the crystal structure of BglB with bound thiocellobiose, a competitive inhibitor, which together with the BglB-cellotetraose complex delineate the general features of the aglycon site. The detailed characterization of the atomic interactions at the aglycon site show a recognition pattern common to all bacterial beta-glucosidases, and presents some differences with the aglycon site in plant beta-glycosidases essentially by means of a different orientation of the basal Trp. The crystal structures of of BglB with a covalently bound inhibitor (derived from 2-fluoroglucoside) and glucose (produced by hydrolysis of the substrate in the crystal), provide additional pictures of the binding events and the intermediates formed during the reaction. Altogether, this information can assist in the understanding of subtle differences of the enzyme mechanism and substrate recognition within this family of enzymes, and consequently it can help in the development of new enzymes with improved activity or specificity.