The specificity of oxidase and kinase preparations from Pseudomonas fluorescens towards deoxyfluoromonosaccharides.

With partially purified enzyme preparations from cell-free extracts of Pseudomonas fluorescens, 3-deoxy-3-fluoro-D-glucose and 3-deoxy-3-fluoro-D-gluconic acid are substrates for glucose oxidase (EC 1.1.3.4.) and gluconate dehydrogenase (EC 1.1.99.3), with K-m values 18.2 mM and 11.8 mM, respectively. The same enzymes that oxidize glucose and gluconic acid probably oxidize 3-deoxy-3-fluoro-D-glucose and 3-deoxy-3-fluoro-D-gluconic acid. The latter fluorinated carbohydrates and the presumed formation of 3-deoxy-3-fluoro-2-keto-D-gluconic acid, which has been isolated as a calcium salt and characterizied, are not substrates for gluconokinase (EC 2.7.1.12). Both 3-deoxy-3-fluoro-D-glucose and 3-deoxy-3-fluoro-D-gluconic acid act as competitive inhibitors of this enzyme preparation for gluconate, with K-i values 47.5 mM and 14.8 mM, respectively.     

The formation of alpha-D-(1----3) branch linkages by a D-glucansucrase from Streptococcus mutans 6715 producing a soluble D-glucan

An exocellular D- glucansucrase that synthesizes a water-soluble, alpha-D-(1----6)-linked D-glucan having a high proportion of alpha-D-(1----3) branches was purified from the culture broth of Streptococcus mutans 6715. The rate of incorporation of D-[14C]glucose from [14C]sucrose into D-glucan of high molecular weight by this enzyme was increased (stimulated) by the presence of exogenous Leuconostoc mesenteroides B- 512F dextran, and it was found that this dextran could act as an acceptor. A highly branched dextran, containing 45-50% of alpha-D-(1----3) branch linkages, did not stimulate the enzyme nearly so much as B- 512F dextran, which has a low degree (5%) of alpha-D-(1----3) branches. We interpret this as evidence that the stimulating effects of dextran are not due to priming. If they were, the more highly branched dextran should have produced the greatest stimulation per unit weight, because a much greater number of nonreducing-end, priming sites would be available. We show that the D- glucansucrase was capable of transferring D-glucosyl groups from sucrose to B- 512F dextran to form alpha-D-(1----3) branches, thereby rendering the dextran more resistant to hydrolysis by endodextranase . The presence of 1.6M ammonium sulfate caused the enzyme to synthesize a D-glucan having a much higher percentage of alpha-D-(1----3) linkages.     

Synthesis of modified tetrasaccharide sequences of N-glycoproteins

The tetrasaccharides O-alpha-D-mannopyranosyl-(1----3)-O-[alpha-D- mannopyranosyl-(1----6)]-O-(4-deoxy-beta-D-lyxo-hexopyranosyl)-(1- ---4)-2- acetamido-2-deoxy-alpha, beta-D-glycopyranose (22) and O-alpha-D-mannopyranosyl-(1----3)-O-[alpha-D-mannopyranosyl-(1----6)]-O- beta-D-talopyranosyl-(1----4)-2-acetamido-2-deoxy-alpha, beta-D- glucopyranose (37), closely related to the tetrasaccharide core structure of N-glycoproteins, were synthesized. Starting with 1,6-anhydro-2,3-di-O-isopropylidene-beta-D-mannopyranose, the glycosyl donors 3,6-di-O-acetyl-2-O-benzyl-2,4-dideoxy-alpha-D-lyxo- hexopyranosyl bromide (10) and 3,6-di-O-acetyl-2,4-di-O-benzyl-alpha-D-talopyranosyl bromide (30), were obtained in good yield. Coupling of 10 or 30 with 1,6-anhydro-2-azido-3-O-benzyl-beta-D-glucopyranose to give, respectively, the disaccharides 1,6-anhydro-2-azido-3-O-benzyl-2-deoxy-4-O-(3,6-di-O-acetyl-2-O-benzyl-4 -deoxy- beta-D-lyxo-hexopyranosyl)-beta-D-glucopyranose and 1,6-anhydro-2-azido-3-O-benzyl-2-deoxy-4-O-(3,6-di-O-acetyl-2,4-di-O-ben zyl- beta-D-talopyranosyl)-beta-D-glucopyranose was achieved with good selectivity by catalysis with silver silicate. Simultaneous glycosylation of OH-3' and OH-6' of the respective disaccharides with 2-O-acetyl-3,4,6-tri-O-benzyl-alpha-D-mannopyranosyl chloride yielded tetrasaccharide derivatives, which were deblocked into the desired tetrasaccharides 22 and 37.     

The synthesis of a series of modified mannotrisaccharides as probes of the enzymes involved in the early stages of mammalian complex N-glycan formation

A series of mannotrisaccharides were synthesized by two distinct chemical pathways as probes of the enzymes involved in the early stages of mammalian complex N-glycan formation. Methyl (alpha-D-mannopyranosyl)-(1-->3)-[(alpha-D-mannopyranosyl)-(1-->6)]-beta-D-mannopyranoside (6) and methyl (2-deoxy-2-fluoro-alpha-D-mannopyranosyl)-(1-->3)-[(2-deoxy-2-fluoro-alpha-D-mannopyranosyl)-(1-->6)]-beta-D-mannopyranoside (8) were rapidly synthesized from unprotected methyl beta-D-mannopyranoside (12). Methyl (2-deoxy-2-fluoro-alpha-D-mannopyranosyl)-(1-->3)-[(alpha-D-mannopyranosyl)-(1-->6)]-beta-D-mannopyranoside (7) and methyl (alpha-D-mannopyranosyl)-(1-->3)-[(2-deoxy-2-fluoro-alpha-D-mannopyranosyl)-(1-->6)]-beta-D-mannopyranoside (9) were synthesized from the common orthogonally protected precursor methyl 2-O-acetyl-4,6-O-benzylidene-beta-D-mannopyranoside (15). The 2-deoxy-2-fluoro substitution common to trisaccharides 7-9 renders these analogues resistant to enzyme action in two distinct ways. Firstly the fluorine serves as a non-nucleophilic isostere for the acceptor hydroxyl in studies with glycosyl transferases GnT-I and GnT-II (7 and 9, respectively). Secondly it should render trisaccharide 8 stable to hydrolysis by the mannosidases Man-II and Man-III by inductive destabilization of their oxocarbenium ion-like transition states. These analogues should be useful for structural studies on these enzymes.     

2-acetamido-2-deoxy-α-D-galactosidases of clostridium perfringens. separation and characterization of an exoglycosidase and an oligosaccharidase

Two distinct 2-acetamido-2-deoxy-α-D-galactosidases have been separated from filtrates of cultured Clostridium perfringens by electrophoresis in 6.5% poly(acryl-amide) gels. One of the enzymes had a mobility of 0.32-0.36 (relative to Bromophenol Blue) and was identified as the exoglycosidase, 2-acetamido-2-deoxy-α-D-galactosidase. It appears to be the same enzyme as that reported in 1972 by McGuire et al. The second of the two ezymes, having a relative mobility of 0.42-0.46, corresponds to the oligosaccharidase reported in 1972 by Huang and Aminoff. The A-specificities of human type-A erythrocytes and of water-soluble glycoproteins having A-activity are both destroyed by incubation with the 2-acetamido-2-deoxy-α-D-galactosidase, but not on incubation with the oligosaccharidase. A concomitant rise in blood-group O(H) activity, as indicated by the use of a lectin from Ulex europeus, occurred in the A-erythrocytes treated with the exoglycosidase 2-acetamido-2-deoxy-α-D-galactosidase.     

Biochemical and structural features of a novel cyclodextrinase from cow rumen metagenome

A novel enzyme, RA.04, belonging to the alpha-amylase family was obtained after expression of metagenomic DNA from rumen fluid (Ferrer et al.: Environ. Microbiol. 2005, 7, 1996-2010). The purified RA.04 has a tetrameric structure (280 kDa) and exhibited maximum activity (5000 U/mg protein) at 70 degrees C and was active within an unusually broad pH range from 5.5 to 9.0. It maintained 80% activity at pH 5.0 and 9.5 and 75 degrees C. The enzyme hydrolyzed alpha-D-(1,4) bonds 13-fold faster than alpha-D-(1,6) bonds to yield maltose and glucose as the main products, and it exhibited transglycosylation activity. Its preferred substrates, in the descending order, were maltooligosaccharides (C3-C7), cyclomaltoheptaose (beta-CD), cyclomaltohexaose (alpha-CD), cyclomaltooctaose (gamma-CD), soluble starch, amylose, pullulan and amylopectin. The biochemical properties and amino acid sequence alignments suggested that this enzyme is a cyclomaltodextrinase. However, despite the similarity in the catalytic module (with Glu359 and Asp331 being the catalytic nucleophile and substrate-binding residues, respectively), the enzyme bears a shorter N-terminal domain that may keep the active site more accessible for both starch and pullulan, compared to the other known CDases. Moreover, RA.04 lacks the well-conserved N-terminal Trp responsible for the substrate preference typical of CDases/MAases/PNases, suggesting a new residue is implicated in the preference for cyclic maltooligosaccharides. This study has demonstrated the usefulness of a metagenomic approach to gain novel debranching enzymes, important for the bread/food industries, from microbial environments with a high rate of plant polymer turnover, exemplified by the cow rumen.     

Crystal structure and stereochemical studies of KD(P)G aldolase from Thermoproteus tenax

Carbon-carbon bond forming enzymes offer great potential for organic biosynthesis. Hence there is an ongoing effort to improve their biocatalytic properties, regarding availability, activity, stability, and substrate specificity and selectivity. Aldolases belong to the class of C-C bond forming enzymes and play important roles in numerous cellular processes. In several hyperthermophilic Archaea the 2-keto-3-deoxy-(6-phospho)-gluconate (KD(P)G) aldolase was identified as a key player in the metabolic pathway. The carbohydrate metabolism of the hyperthermophilic Crenarchaeote Thermoproteus tenax, for example, has been found to employ a combination of a variant of the Embden-Meyerhof-Parnas pathway and an unusual branched Entner-Doudoroff pathway that harbors a nonphosphorylative and a semiphosphorylative branch. The KD(P)G aldolase catalyzes the reversible cleavage of 2-keto-3-deoxy-6-phosphogluconate (KDPG) and 2-keto-3-deoxygluconate (KDG) forming pyruvate and glyceraldehyde 3-phosphate or glyceraldehyde, respectively. In T. tenax initial studies revealed that the pathway is specific for glucose, whereas in the thermoacidophilic Crenarchaeote Sulfolobus solfataricus the pathway was shown to be promiscuous for glucose and galactose degradation. The KD(P)G aldolase of S. solfataricus lacks stereo control and displays additional activity with 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) and 2-keto-3-deoxygalactonate (KDGal), similar to the KD(P)G aldolase of Sulfolobus acidocaldarius. To address the stereo control of the T. tenax enzyme the formation of the two C4 epimers KDG and KDGal was analyzed via gas chromatography combined with mass spectroscopy. Furthermore, the crystal structure of the apoprotein was determined to a resolution of 2.0 A, and the crystal structure of the protein covalently linked to a pathway intermediate, namely pyruvate, was determined to 2.2 A. Interestingly, although the pathway seems to be specific for glucose in T. tenax the enzyme apparently also lacks stereo control, suggesting that the enzyme is a trade-off between required catabolic flexibility needed for the conversion of phosphorylated and nonphosphorylated substrates and required stereo control of cellular/physiological enzymatic reactions.     

Improving upon nature: active site remodeling produces highly efficient aldolase activity toward hydrophobic electrophilic substrates

The substrate specificity of enzymes is frequently narrow and constrained by multiple interactions, limiting the use of natural enzymes in biocatalytic applications. Aldolases have important synthetic applications, but the usefulness of these enzymes is hampered by their narrow reactivity profile with unnatural substrates. To explore the determinants of substrate selectivity and alter the specificity of Escherichia coli 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, we employed structure-based mutagenesis coupled with library screening of mutant enzymes localized to the bacterial periplasm. We identified two active site mutations (T161S and S184L) that work additively to enhance the substrate specificity of this aldolase to include catalysis of retro-aldol cleavage of (4S)-2-keto-4-hydroxy-4-(2'-pyridyl)butyrate (S-KHPB). These mutations improve the value of k(cat)/K(M)(S-KHPB) by >450-fold, resulting in a catalytic efficiency that is comparable to that of the wild-type enzyme with the natural substrate while retaining high stereoselectivity. Moreover, the value of k(cat)(S-KHPB) for this mutant enzyme, a parameter critical for biocatalytic applications, is 3-fold higher than the maximal value achieved by the natural aldolase with any substrate. This mutant also possesses high catalytic efficiency for the retro-aldol cleavage of the natural substrate, KDPG, and a >50-fold improved activity for cleavage of 2-keto-4-hydroxy-octonoate, a nonfunctionalized hydrophobic analogue. These data suggest a substrate binding mode that illuminates the origin of facial selectivity in aldol addition reactions catalyzed by KDPG and 2-keto-3-deoxy-6-phosphogalactonate aldolases. Furthermore, targeting mutations to the active site provides a marked improvement in substrate selectivity, demonstrating that structure-guided active site mutagenesis combined with selection techniques can efficiently identify proteins with characteristics that compare favorably to those of naturally occurring enzymes.     

Flavobacterium heparinum 3-O-sulphatase for N-substituted glucosamine 3-O-sulphate

A novel bacterial sulphatase has been discovered in an extract of Flavobacterium heparinum. The enzyme hydrolyses the 3-O-sulphate from 2-deoxy-2-sulphamido-3-O-sulpho-D-glucose and 2-acetamido-2-deoxy-3-O-sulpho-D-glucose. The activity was purified 10 800-fold by chromatography successively on CM-Sepharose CL-6B, hydroxyapatite, taurine-Sepharose CL-4B and CM-Sepharose CL-6B. Sodium dodecylsulphate/polyacrylamide gel electrophoresis showed the enzyme to be homogeneous and of relative molecular mass 56 000. Two novel assays were developed using 2-[14C]acetamido-2-deoxy-3-O-sulpho-D-glucose and 2-deoxy-2-sulphamido-3-O-sulpho-D-glucose as respective substrates. The purified 3-O-sulphatase was shown to be free of all other known heparin-degrading enzymes. Optimal activity was at pH 7.5 for the disulphated substrate and pH 8.0 for the N-acetylated substrate. Enzyme activity was virtually unaffected by Na+, K+ or Mg2+ ions. A 1.2-fold enhancement of activity was effected by 0.002 mol dm-3 Ca2+. Inorganic phosphate and sulphate inhibited 3-O-sulphatase activity. The Km value of the N-acetylated substrate was determined to be 42 mumol dm-3. No activity was detected with 2-amino-2-deoxy-3-O-sulpho-D-glucose.     

Heterotrophic Metabolism of the Chemolithotroph Thiobacillus ferrooxidans

Glucose-6-phosphate dehydrogenase and the enzymes of the Entner-Doudoroff pathway, 6-phosphogluconate dehydrase and 2-keto-3-deoxy-6-phosphogluconate aldolase (assayed together), are induced during heterotrophic growth of Thiobacillus ferrooxidans on an iron-glucose-supplemented medium or on glucose alone. By contrast, autotrophic cells (iron-grown) contain low levels of these enzymes. Fructose 1, 6-diphosphate aldolase, an enzyme of the Embden-Meyerhof pathway, is present at low levels irrespective of the growth medium, suggesting that this enzyme is not involved in energy-yielding reactions but merely provides intermediates for biosynthesis. The Entner-Doudoroff and pentose-phosphate pathways are the principle means through which glucose is dissimilated and is presumed to be concerned with energy production. Isotopic studies showed that a high rate of CO(2) formation from specifically labeled glucose came from carbon atoms 1 and 4. An unexpectedly high rate of evolution of CO(2) also came from carbon 6, suggesting that the triose phosphate formed during glucose breakdown and specifically as a result of 2-keto-3-deoxy-6-phosphogluconate aldolase activity, was metabolized via some unorthodox metabolic route. Cells grown in the iron-supplemented and glucose-salts media have a complete tricarboxylic acid cycle, whereas autotrophically grown T. ferrooxidans lacked both alpha-ketoglutarate dehydrogenase and reduced nicotinamide adenine dinucleotide oxidase. Two isocitrate dehydrogenases [nicotinamide adenine dinucleotide (NAD) and NAD phosphate (NADP) specific] were present. NAD-linked enzyme was constitutive, whereas the NADP-linked enzyme was induced upon adaptation of autotrophic cells to heterotrophic growth.     

A chemoenzymatic route to mannosamine derivatives bearing different N-acyl groups

The chemoenzymatic route to 2-deoxy-2-propionamido-D-mannose (1b), 2-butyramido-2-deoxy-D-mannose (2b) and 2-deoxy-2-phenylacetamido-D-mannose (3b) involved N-acylation of 2-amino-2-deoxy-D-glucose followed by alkaline C-2 epimerization and selective microbial removal of the epimers with gluco-configuration. The latter step employed whole cells of Rhodococcus equi A4 able to degrade 2-deoxy-2-propionamido-D-glucose (1a), 2-butyramido-2-deoxy-D-glucose (2a) and 2-deoxy-2-phenylacetamido-D-glucose (3a) but inactive towards the corresponding manno-isomers. The metabolism of the gluco-isomers probably involved phosphorylation and subsequent deacylation. 2-Acetamido-2-deoxy-6-O-phospho-D-glucose amidohydrolase [EC 3.5.1.25] but not 2-acetamido-2-deoxy-D-glucose amidohydrolase was detected in the cell extract, the former enzyme being partially purified (15.8-fold with an overall yield of 18.1% and a specific activity of 0.95 units mg-1 protein). According to SDS-PAGE electrophoresis, gel filtration and mass spectrometry, the enzyme was a monomer with an apparent molecular mass of approximately 42 kDa. The optimum temperature and pH of the enzyme were 60 degrees C and 8.0-9.0, respectively. 2-Acetamido-2-deoxy-6-O-phospho-D-glucose and 2-acetamido-2-deoxy-6-O-sulfo-D-glucose but not 2-acetamido-2-deoxy-1-O-phospho-D-glucose or 2-acetamido-2-deoxy-D-glucose were substrates of the enzyme. Its activity was slightly inhibited by the addition of 1 mM Al3+, Ca2+, Co2+, Cu2+, Mn2+ or Zn2+ and activated by 1 mM Mg2+. The concentrated enzyme is highly stable at 4 degrees C in the presence of 0.1 M ammonium sulfate.     

Studies on cellulolytic enzymes I: The use of 3,4-dinitrophenyl glycosides as substrates

The syntheses of 3,4-dinitrophenyl β-d-glucoside, β-cellobioside, β-cellotrioside, and β-cellotetraoside and their use to monitor the purification of two enzymes from a crude commercial cellulase preparation from Trichoderma viride are described. The enzymes isolated are an endo-β-1,4-d-glucan glucanohydrolase (EI) of molecular weight ca. 12 000 which catalysed the release of 3,4-dinitrophenol from 3,4-dinitrophenol-β-cellotetraoside, and an enzyme of molecular weight about 76 000 which catalysed the hydrolysis of 3,4-dinitrophenyl β-d-glucoside (EII) and is probably a cellobiase or exo-β-1,4-d-glucan glucohydrolase. Kinetic parameters are reported for the hydrolyses of 3,4-dinitrophenyl β-cellobioside, β-cellotrioside, and β-cellotetraoside catalysed by enzyme EI. In the presence of cellotriose, cellotetraose, or cellopentaose 3,4-dinitrophenyl β-d-glucoside underwent induced hydrolyses by EI. Similar but faster induced hydrolyses were shown by 3,4-dinitrophenyl β-d-xyloside and 3,4-dinitrophenyl β-d-6-deoxyglucoside; 3,4-dinitrophenyl 6-chloro-6-deoxy-β-d-glucoside and 3,4-dinitrophenyl 6-O-methyl-β-d-glucoside underwent slower induced hydrolyses than the glucoside. p-Nitrophenyl β-d-glucoside also underwent an induced hydrolysis in the presence of cellopentaose and the enzyme EI, but p-nitrophenyl 2-deoxy-β-d-glucoside did not. These results are discussed and compared with the results obtained previously on induced hydrolyses found with lysozyme. Kinetic parameters are reported for the hydrolysis of 3,4-dinitrophenyl and p-nitrophenyl β-d-glucosides catalysed by the enzyme EII. 3,4-Dinitrophenyl 6-deoxy-β-d-glucoside, β-d-xyloside, 6-chloro-6-deoxy-β-d-glucoside, 6-O-methyl-β-d-glucoside and p-nitrophenyl-β-d-galactopyranoside and 2-deoxy-β-d-glucopyranoside were hydrolysed 10² to 10³ times slower by EII than the corresponding glucosides, but 3,4-dinitrophenyl 2-acetamido-2-deoxy-β-d-glucoside was only hydrolysed about 25 times slower than 3,4-dinitrophenyl β-d-glucoside. The significance of these results is discussed. EII catalysed the release of 3,4-dinitrophenol from 3,4-dinitrophenyl β-cellobioside, β-cellobioside, and β-cellotetraoside, but these reactions showed induction periods which are consistent with stepwise removal of glucose residues from the oligosaccharide chains before release of the phenol.     

Improved functional properties of trypsin modified by monosubstituted amino-β-cyclodextrins

Bovine pancreatic trypsin was chemically modified by several β-cyclodextrin (β-CD) derivatives using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as coupling agent. The modifying agents used were mono-6-amino-6-deoxy-β-cyclodextrin (CDNH₂), mono-6-ethylenediamino-6-deoxy-β-cyclodextrin (CDEN), mono-6-propylenediamino-6-deoxy-β-cyclodextrin (CDPN) and mono-6-butylenediamino-6-deoxy-β-cyclodextrin (CDBN). The enzyme–cyclodextrin conjugates contained about 2 mol of oligosaccharide per mol of trypsin. The catalytic and thermal stability properties of trypsin were improved by the attachment of cyclodextrin residues, and these effects were markedly noticeable for cyclodextrin (CD) derivatives having an even number of carbon atoms in the spacer arms. The thermostability of the enzyme was increased by about 2.4–14.5 °C after modification. The conjugates prepared were also more stable against thermal incubation at different temperatures ranging from 45 to 60 °C. In comparison with native trypsin, the enzyme–cyclodextrin complexes were markedly more resistant to autolytic degradation at pH 9.0. Attending to the results here reported, we suggest that conjugation of enzymes with β-CD derivatives might be an useful method for improving the stability and the catalytic properties of these biocatalysts.     

Purification of heparinase and heparitinase by affinity chromatography on glycosaminoglycan-bound ah-sepha-rose 4b

Heparinase and heparitinase were separated from an extract of Flavobacterium heparinum, induced with heparin by using column chromatography on hydroxylapatite. As the heparinase preparation contained chondroitinases B and C, chondroitinase B was removed by rechromatography on a hydroxylapatite column. Chondroitinase C was then eliminated by column chromatography on O-phosphono(“phospho”)-cellulose. The heparinase preparation thus obtained was free from sulfoamidase for 2-deoxy-2-sulfoamino-D-glucose (GlcN-2S), sulfatase for 2-amino-2-deoxy-6-O-sulfo D-glucose (GlcN-6S), as well as (δ_4,5glycosiduronase for the unsaturated disaccharides obtained from heparin. The remaining sulfatase for 4-deoxy-α-L-thero-hex-4-enopyranosyluronic acid 2-sulfate (δUA-2S) in the heparinase preparation was removed by affinity chromatography with dermatan sulfate-bound AH-Sepharose 4B coated with dermatan sulfate. The heparitinase preparation separated by column chromatography on hydroxyla patite was purified by affinity chromatography with heparin-bound AH-Sepharose 4B coated with heparin. Sulfatase for 2-amino-2-deoxy-6-O-sulfo-D-glucose (GlcN-6S) and δ_4,5glycosiduronase for the unsaturated disaccharides obtained from heparin were removed by this chromatography. Sulfatase for 4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid 2-sulfate (δUA-2S) remaining in the heparitinase preparation was finally removed by column chromatography on hydroxylapatite. The recoveries of the purified preparations of heparinase and heparitinase were estimated to be 39 and 50%, respectively, from the crude enzyme fractions obtained by the first column chromatography on hydroxyl- patite. The purified heparinase and heparitinase were free from all enzymes that could degrade the sulfated unsaturated disaccharides produced from heparin with heparinase.     

A biochemical study of the intermediary carbon metabolism of Shewanella putrefaciens.

Cell extracts were used to determine the enzymes involved in the intermediary carbon metabolism of several strains of Shewanella putrefaciens. Enzymes of the Entner-Doudoroff pathway (6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase) were detected, but those of the Embden-Meyerhof-Parnas pathway were not. While several tricarboxylic acid cycle enzymes were present under both aerobic and anaerobic conditions, two key enzymes (2-oxoglutarate dehydrogenase and pyruvate dehydrogenase) were greatly diminished under anaerobic conditions. Extracts of cell grown anaerobically on formate as the sole source of carbon and energy were positive for hydroxypyruvate reductase, the key enzyme of the serine pathway in other methylotrophs, while no hexulose synthase activity was seen.     

Syntheses of model compounds related to an antigenic epitope in pectic polysaccharides from Bupleurum falcatum L

The beta-galactosidases from Xanthomonas manihotis (beta-Gal Xmn) and Bacillus circulans (beta-Gal-3 Bcir) are retaining glycosidases that hydrolyze glycosidic bonds through a double displacement mechanism involving a covalent glycosyl-enzyme intermediate. The mechanism-based inactivator 2,4-dinitrophenyl 2-deoxy-2-fluoro-beta-D-galactopyranoside was shown to inactivate beta-Gal Xmn and beta-Gal-3 Bcir through the accumulation of 2-deoxy-2-fluorogalactosyl enzyme intermediates with half lives of 40 and 625 h, respectively. Peptic digestion of these labeled enzymes and analysis by LC-MS identified Glu(260) and Glu(233) as the catalytic nucleophiles involved in the formation of the glycosyl-enzyme intermediate during catalysis by beta-Gal Xmn and beta-Gal-3 Bcir, respectively. These findings confirm the previous prediction of the position of these residues based on primary sequence similarities to other members of the glycoside hydrolase family 35.     

New substrate specificity of modified porcine pancreatic alpha-amylase

Conversion of the substrate specificity of porcine pancreatic alpha-amylase (PPA) was studied using chemical modification of His residues. Diethyl pyrocarbonate modified His residues in PPA and the activity of the modified PPA for the hydrolysis of the alpha-D-(1,4)glucoside bond in starch or oligosaccharides decreased to less than 1% of that of the native enzyme. However, the activity for the hydrolysis of the bond between p-nitrophenol and oligosaccharides in p-nitrophenyl oligosaccharides was increased by chemical modification. When the modified PPA was incubated with a proteinaceous alpha-amylase inhibitor (Mr 60,000) purified from white kidney bean (Phaseolus vulgaris), it bound to the inhibitor. As a result, the remaining less than 1% hydrolytic activity of the modified PPA for starch disappeared completely but that for p-nitrophenyl oligosaccharides remained unaltered. The hydrolytic activity of the native PPA for the alpha-D-(1,4)glucoside bond in oligosaccharides was stronger than that between p-nitrophenyl and oligosaccharides in p-nitrophenyl oligosaccharides. Therefore, when p-nitrophenyl oligosaccharides (three to five glucose residues) were used as substrates for the native PPA, the alpha-D-(1,4)glucoside bonds in the oligosaccharides were hydrolyzed. However, the modified PPA-inhibitor complex hydrolyzed only the bond between p-nitrophenol and oligosaccharides in p-nitrophenyl oligosaccharides. The above results reveal that, by chemical modification with diethyl pyrocarbonate and biochemical modification with an amylase inhibitor, amylase can be converted to a new exo-type enzyme which hydrolyzes only the bond between p-nitrophenol and oligosaccharides in p-nitrophenyl oligosaccharides.     

Human acid beta-glucosidase: inhibition studies using glucose analogues and pH variation to characterize the normal and Gaucher disease glycon binding sites

Comparative kinetic studies with glycon inhibitors were used to investigate the properties of the active site of human acid beta-glucosidase (EC 3.2.1.45) from normal placenta and spleens of type 1 Ashkenazi Jewish Gaucher disease (AJGD) patients. With the pure normal enzyme, the specificity of glycon binding was assessed with 35 glucose derivatives and epimers. Most glycons were mixed type inhibitors with a predominantly competitive nature (i.e., Kis much less than Kii) and had low apparent affinity for the enzyme (Kisapp = 20-500 mmol/l). beta-Glucose-1-phosphate was unusual, since it inhibited 4-methylumbelliferyl-beta-glucoside hydrolysis in an uncompetitive pattern (Kiapp = 0.55 mmol/l) but had no effect on glucosyl ceramide hydrolysis. C-1- (1-deoxy-1-amino-beta-D-glucose) and C-3- (3-deoxy-3-amino-D-glucose) amino and C-5-imino [1-deoxynojirimycin (dNM), nojirimycin and castanospermine] substituted sugars were highly potent inhibitors with Kisapp(beta-glucose)/Kisapp approximately equal to 10(3)-10(5); an amine at C-2 did not alter Kisapp compared to beta-glucose. The variation of Kisapp with pH for the 5-imino- and 1-deoxy-1-aminoglycosides conformed to a model for the unprotonated inhibitors binding to the protonated forms (EH and EH2) of the diprotic (Vmaxapp and Vmaxapp/Kmapp) normal enzyme (pK1 = 4.7; pK2 = 6.7) with pH-independent Kisapp values of 2.9-9.0 mumol/l and 0.22 mmol/l, respectively. Several of the amine-containing inhibitors competitively protected the enzyme from inactivation by conduritol B epoxide, a covalent active site-directed inhibitor, indicating interaction with residues at that site. With the partially purified AJGD splenic enzymes, the results were the same except that Kisapp(AJGD)/Kisapp(normal) = 4-17 for dNM and 1-deoxy-1-amino-beta-glucose; this ratio was approximately equal to 1 with most other glycons, and particularly, nojirimycin and castanospermine. The results of these studies indicated that the glycon binding site of the normal acid beta-glucosidase contains important residues for interaction with the C-2, C-3 and C-4 hydroxyl groups of beta-glucose and a residue with pKa = 6.7 which was critical to the binding of amine-containing inhibitors and the hydrolysis of substrates. The findings were consistent with a specific alteration in or near the glycon binding site which results in the functional abnormalities of the mutant AJGD acid beta-glucosidase.     

Identification of the catalytic nucleophile of the family 29 alpha-L-fucosidase from Thermotoga maritima through trapping of a covalent glycosyl-enzyme intermediate and mutagenesis

Fucose-containing glycoconjugates are key antigenic determinants in many biological processes. A change in expression levels of the enzymes responsible for tailoring these glycoconjugates has been associated with many pathological conditions and it is therefore surprising that little information is known regarding the mechanism of action of these important catabolic enzymes. Thermotoga maritima, a thermophilic bacterium, produces a wide range of carbohydrate-processing enzymes including a 52-kDa alpha-L-fucosidase that has 38% sequence identity and 56% similarity to human fucosidases. The catalytic nucleophile of this enzyme was identified to be Asp-224 within the peptide sequence 222WNDMGWPEKGKEDL235 using the mechanism-based covalent inactivator 2-deoxy-2-fluoro-alpha-L-fucosyl fluoride. The 10(4)-fold lower activity (kcat/Km) of the site-directed mutant D224A, and the subsequent rescue of activity upon addition of exogenous nucleophiles, conclusively confirms this assignment. This article presents the first direct identification of the catalytic nucleophile of an alpha-L-fucosidase, a key step in the understanding of these important enzymes.     

Effect of blood plasma inhibitors on activity of serine and cysteine proteinases]

Data on study of action plasma inhibitors on activity of pancreatic proteolytic enzymes (trypsin, chymotrypsin) and plant proteinases (papain, bromelain), included in composition of enzyme mixes, used for orally application are submitted. It is established, that serine proteases are more sensitive to inactivation of plasma inhibitors, than cysteine enzymes. Main inhibitor of the papain and bromelain is alpha-2-macroglobulin in complex with which they preserve significant part of initial activity. A high-sensitivity method of determination of activity enzyme combinations, enabling to detect nanograms of them in presence of plasma inhibitors is offered. It can be used for study pharmacokinetic and optimization of enzyme mixes application in clinical practice.