Disaccharide Substrates Research Articles

Purification and Properties of a Cellobiose Phosphorylase (CepA) and a Cellodextrin Phosphorylase (CepB) from the Cellulolytic Thermophile Clostridium Stercorarium

Two phosphorolytic enzymes displaying activity towards the soluble cellulose degradation products cellobiose and cellodextrins were purified from the crude extract of the cellulolytic thermophile Clostridium stercorarium. Both phosphorylases have monomeric structures with molecular masses of 93 and 91 kDa, respectively. Although the N-terminal amino acid sequences are highly similar, a clear distinction of the two enzymes could be made on the basis of their substrate specificities: the enzyme designated cellobiose phosphorylase cleaved exclusively the disaccharide substrate, whereas the enzyme designated cellodextrin phosphorylase accepted only oligosaccharides as substrates. Kinetic constants were determined for the cleavage of cellobiose and cellodextrins. Maximal activity was observed at 65 degrees C in the pH range 6.0-7.0 for both enzymes. The sequences of the genes cepA and cepB encoding the cellobiose phosphorylase and the cellodextrin phosphorylase, respectively, have been submitted to the GenBank database.

Structure-Function Analysis of Human 1,3-Fucosyltransferase: AMINO ACIDS INVOLVED IN ACCEPTOR SUBSTRATE SPECIFICITY

A series of molecular biology experiments were carried out to identify the catalytic domain of two human alpha1,3/4-fucosyltransferases (fucosyltransferases (FucTs) III and V), and to identify amino acids that function in acceptor substrate binding. Sixty-one and 75 amino acids could be eliminated from the N terminus of FucTs III and V, respectively, without a significant loss of enzyme activity. In contrast, the truncation of one or more amino acids from the C terminus of FucT V resulted in a dramatic or total loss of enzyme activity. Results from the truncation experiments demonstrate that FucT III62-361 (containing amino acids 62-361) and FucT V76-374 (containing amino acids 76-374) are active, whereas shorter forms of the enzymes were inactive. The shortest, active forms of the enzymes are more than 93% identical at the predicted amino acid level, but have distinct acceptor substrate specificities. Thus, FucT III is an alpha1,4-fucosyltransferase, whereas FucT V is an alpha1,3-fucosyltransferase with disaccharide substrates. All but one of the amino acid sequence differences between the two proteins occur near their N terminus. Results obtained from domain swapping experiments demonstrated that the single amino acid sequence difference near the C terminus of these enzymes did not alter the enzyme's substrate specificity. However, swapping a region near the N terminus of the truncated form of FucT III into an homologous region in FucT V produced a protein with both alpha1,3- and alpha1,4-fucosyltransferase activity. This region contains 8 of the amino acid sequence differences that occur between the two proteins.

Human acetyl-coenzyme A:alpha-glucosaminide N-acetyltransferase. Kinetic characterization and mechanistic interpretation.

Acetyl-CoA: alpha-glucosaminide N-acetyltransferase (N-acetyltransferase) is an integral lysosomal membrane protein which catalyses the transfer of acetyl groups from acetyl-CoA on to the terminal glucosamine in heparin and heparan sulphate chains within the lysosome. In vitro, the enzyme is capable of acetylating a number of mono- and oligo-saccharides derived from heparin, provided that a non-reducing terminal glucosamine is present. We have prepared highly enriched lysosomal membrane fractions from human placenta by a combination of differential centrifugation and density-gradient centrifugation in Percoll. This preparation was used to investigate the kinetics of the enzyme with three acetyl-acceptor substrates, i.e. glucosamine and a disaccharide and a tetrasaccharide derived from heparin, each containing a terminal glucosamine residue. The enzyme showed a pH optimum at 6.5, extending to 8.0 for the mono- and di-saccharide substrates but falling off sharply above pH 6.5 for the tetrasaccharide substrate. We identified two distinct Km values for the glucosamine substrate at both pH 7.0 and pH 5.0, whereas the tetrasaccharide substrate displayed only a single Km value at each pH. The Km values were found to be highly pH-dependent, and at pH 5.0 the values for the acetyl-acceptor substrates showed a decreasing trend as the size of the substrate increased, suggesting that the enzyme recognizes an extended region of the non-reducing terminus of the heparin or heparan sulphate polysaccharides. Double-reciprocal analysis, isotope exchange between N-acetylglucosamine and glucosamine, and inhibition studies with desulpho-CoA indicate that the enzyme operates by a random-order ternary-complex mechanism. Product inhibition studies display a complex pattern of dead-end inhibition. Taken in context with what is known about lysosomal utilization and physiological levels of acetyl-CoA, these results suggest that in vivo the enzyme operates via a random-order ternary-complex mechanism which involves the utilization of cytosolic acetyl-CoA to transfer acetyl groups on to the terminal glucosamine residues of heparin within the lysosome.

Oligosaccharyltransferase: synthesis and use of deuterium-labeled peptide substrates as mechanistic probes.

Chemically synthesized peptide and lipid disaccharide substrates have been used to investigate two possible mechanisms for enzyme-catalyzed N-glycosylation. Using microsomal oligosaccharyltransferase isolated from yeast, the fate of the deuterium in three stereospecifically deuterated peptides has been investigated. In all three cases, the deuterium present in the peptide substrate was retained in the glycopeptide product, as shown clearly by 1H NMR spectral comparisons. The lack of deuterium wash-out during catalysis provides strong evidence against either enol lactone or ketene formation as an intermediate in this reaction.

Correction. Enzyme-Catalyzed Glycosylation of Peptides Using a Synthetic Lipid Disaccharide Substrate

Correction. Enzyme-Catalyzed Glycosylation of Peptides Using a Synthetic Lipid Disaccharide Substrate

Enzyme-catalyzed glycosylation of peptides using a synthetic lipid disaccharide substrate

A lipid disaccharide, consisting of chitobiose linked to dolichol via an α-1-pyrophosphate, has been synthesized for use as a substrate in the enzyme-catalyzed glycosylation of peptides. For the purpose of confirming the structure of the reaction product, the expected glycopeptide was synthetized via an unambiguous, convergent method. Chromatographic and spectral comparison of the synthetic vs synthetic glycopeptide showed that they were identical

Hurler syndrome: A patient with abnormally high levels of α-l-iduronidase protein

Mucopolysaccharidosis type I (MPS I: McKusick 25280) is a clinically heterogenous lysosomal storage disorder which is caused by a variable deficiency in alpha-L-iduronidase activity (alpha-L-iduronide iduronohydrolase, EC 3.2.1.76). Cultured fibroblasts from an MPS I patient (cell line 2827) with a severe clinical phenotype (Hurler syndrome) have been characterized using immunochemical and biochemical techniques. Using a specific immunoquantification assay, we have demonstrated that cell line 2827 had an alpha-L-iduronidase protein content (189 ng/mg of extracted cell protein) at least six times greater than the mean level found in normal control fibroblasts (30 ng/mg of extracted cell protein). This was the only MPS I cell line, from a group of 23 MPS I patients, that contained greater than 7% of the mean level of alpha-L-iduronidase protein detected in normal controls. Cell line 2827 had very low alpha-L-iduronidase activity toward the fluorogenic substrate 4-methylumbelliferyl-alpha-L-iduronide, and a radiolabeled disaccharide substrate derived from heparin. Maturation studies of alpha-L-iduronidase in cell line 2827 showed apparently normal levels of alpha-L-iduronidase synthesis with delayed processing to the mature form. Subcellular fractionation experiments demonstrated alpha-L-iduronidase protein in lysosomal-enriched fractions isolated from cell line 2827, suggesting a normal cell distribution and supporting the proposed delayed processing. It is proposed that the MPS I patient described has an alpha-L-iduronidase gene mutation which affects both the active site and post-translational processing of the enzyme. This mutation must be structurally conservative because it does not result in instability either during maturation or in the lysosome.

Roles of the aromatic side chains in the binding of substrates, inhibitors, and cyclomalto-oligosaccharides to the glucoamylase from Aspergillus niger probed by perturbation difference spectroscopy, chemical modification, and mutagenesis

The roles of the aromatic side chains of the glucoamylase from Aspergillus niger in the binding of ligands, as determined by differences spectroscopy using four types of inhibitors ( a) valienamine-derived, ( b) 1-deoxynojrimyscins, ( c) d-glucono-1,5-lactone, and ( d), two types of disaccharide substrates ( a) α-(1»4)-linked and ( b) α-(1»6)-linked, and three cyclomalto-oligosaccharides (cyclodextrins, CDs) are discussed. An unusual change in absorbance from 300 to 310—320 nm, obtained only with valienamine-derived inhibitors or when d-glucono-1,5-lactone and maltose are combined, is concluded to arise when subsite 2 is occupied in a transition-state-type of complex. The single mutations of two residues thought to be involved in binding, namely, Tyr116»Ala and Trp120»Phe, alter, but do not abolish this perturbation. The perturbations in the spectra also suggest that maltose and isomaltose have different modes of binding. The following K d values ( m) were determined: acarbose, < x 10 -12; methyl acarviosinide, 1.6 x 10 -6; and the d- gluco and l- ido forms of hydrogenated acarbose, 1.4 x 10 -8 and 5.2 x 10 -6, respectively. Therefore, both the valienamine moiety and the chain length of acarbose are important for tight binding. In contrast to the valienamine-derived inhibitors, none of the 1-deoxynojirimycin type protected glucoamylase against inactivating oxidation of tryptophyanyl residues, although each had a K d value of ∼ 4 x 10 -6 m. There are two distinct carbohydrate-binding areas in glucoamylase, namely, the active site in the catalytic domain and a strach-granule-binding site in the C-terminal domain. The α-,β-, and γ-CDs have high affinity for the starch-binding domain and low affinity for the active site, whereas the reverse was found for acarbose.

Human alpha-L-iduronidase. Catalytic properties and an integrated role in the lysosomal degradation of heparan sulphate.

The kinetic parameters (Km and kcat) of human liver alpha-L-iduronidase were determined with a variety of heparin-derived disaccharide and tetrasaccharide substrates. More structurally complex substrates, in which several aspects of the aglycone structure of the natural substrates heparin and heparan sulphate were maintained, were hydrolysed with catalytic efficiencies up to 255 times that observed for the simplest disaccharide substrate to be hydrolysed. The major aglycone structure that influenced both substrate binding and enzyme activity was the presence of a C-6 sulphate ester on the residue adjacent to the iduronic acid residue being hydrolysed. Sulphate ions and a number of substrate and product analogues were potent inhibitors of enzyme activity. Human liver alpha-L-iduronidase activity towards 4-methylumbelliferyl alpha-L-iduronide at pH 4.8 had two Km values of 37 microM and 1.92 mM with corresponding kcat. values of 299 and 650 mol of product formed/min per mol of enzyme respectively, which may explain the wide range of Km values previously reported for alpha-L-iduronidase activity toward its substrate. Skin fibroblast alpha-L-iduronidase activity towards the heparin-derived oligosaccharides was influenced by the same substrate aglycone structural features as was observed for the human liver enzyme. A comparison was made of the effect of substrate aglycone structure upon catalytic activities of the enzymes which act to degrade the highly sulphated regions of heparan sulphate. A model was proposed whereby the substrate is directed from alpha-L-iduronidase to subsequent enzyme activities to ensure the efficient degradation of heparan sulphate.

Glucuronate-2-sulphatase activity in cultured human skin fibroblast homogenates

The optimization of the assay conditions to detect glucuronate-2-sulphatase (GS) activity present in cultured human skin fibroblast homogenates towards a heparin-derived disaccharide substrate O-(beta-D-glucuronic acid 2-sulphate)-(1----4)-D-O-2,5-anhydro[l-3H]mannitol 6-sulphate (GSMS) has shown that a complex relationship exists between pH, buffer composition, ionic strength and the influence of added BSA and salts (NaCl, Na2SO4, CuCl2 and ZnCl2) to achieve maximum sulphatase activity. Whereas albumin stimulated GS activity by more than 2-fold over the pH range 2.7-5.7, CuCl2 stimulated GS activity over the narrow pH range 3.0-4.2, and inhibited GS activity at higher pH. ZnCl2 stimulated GS activity more than 3-fold at pH 3.0 and by more than 10-fold at pH 4.8. NaCl inhibited GS activity at pH 3.0, while activity between pH 4.2 and 4.8 was stimulated by up to 10-fold, resulting in a shift in the observed pH optimum from 3.0 to 4.8 in the presence of 315 mM-NaCl. Skin fibroblast GS activity toward GSMS had apparent Km values of 0.5-1.2 microM at pH 3.0, and 27.0-33.2 microM at pH 4.8. Albumin stimulated GS activity at both low and high pH by an increase in the apparent Vmax. values without significant alteration in the respective Km values. At pH 4.8, NaCl stimulated GS activity as a result of an increase in Vmax. values. These observations raise the possibility that two forms of GS activity are present in skin fibroblast homogenates: a low-Km form that has a pH optimum of 3.0 and is stimulated by BSA and a high-Km form with a pH optimum of 4.8 which is stimulated by NaCl.

Purification and characterization of GDP-L-Fuc-N-acetyl-beta-D-glucosaminide alpha 1—-3fucosyltransferase from human neuroblastoma cells. Unusual substrate specificities of the tumor enzyme

Fucosyl residues in the alpha 1----3 linkage to N-acetylglucosamine (Fuc alpha 1----3GlcNAc) on oligosaccharides of glycoproteins and glycolipids have been detected in certain human tumors and are developmentally expressed (reviewed in Foster, C. S., and Glick, M. C. (1988) Adv. Neuroblastoma Res. 2, 421-432). In order to understand control mechanisms for the biosynthesis of these fucosylated glycoconjugates, GDP-L-Fuc-N-acetyl-beta-D-glucosaminide alpha 1----3fucosyltransferase was purified from human neuroblastoma cells, CHP 134, utilizing either the immobilized oligosaccharide or disaccharide substrates. The enzyme, extracted from CHP 134 cells, was purified by DEAE- and SP-Sephadex chromatography and then by either immobilized substrate. alpha 1----3Fucosyltransferase was obtained in approximately 10% yield and was purified 45,000-fold from the cell extract. The kinetic properties of the enzyme showed an apparent KGDP-Fuc 43 microM, KGal beta 1----4GlcNAc 0.4 mM, KGal beta 1----4Glc 8.1 mM, and KFuc alpha 1----2Gal beta 1----4Glc 1.0 mM. Polyacrylamide gel electrophoresis of the affinity-purified enzyme showed two proteins which migrated, Mr = 45,000-40,000. The enzyme differed in substrate specificity, pH optimum, response to N-ethylmaleimide and ion requirements from the enzymes purified from human milk or serum. The inability of alpha 1----3fucosyltransferase to transfer to substrates containing NeuAc alpha 2----3 or alpha 2----6Gal is in contrast to the reports for the enzyme in other human tumors. This substrate specificity correlates with the oligosaccharide residues thus far defined on glycoproteins of CHP 134 cells since NeuAc and Fuc alpha 1----3GlcNAc have yet to be detected on the same oligosaccharide antenna. However, the enzyme transfers to Fuc alpha 1----2Gal beta 1----4GlcNAc/Glc with higher activity than the unfucosylated disaccharides, although neither alpha 1----2fucosyltransferase nor Fuc alpha 1----2 residues have been detected in CHP 134 cells. The different substrate specificities of alpha 1----3fucosyltransferase isolated from human tumors and normal sources leads to the suggestion that a family of alpha 1----3fucosyltransferases may exist and that they may be differentially expressed in human tumors.

Human liver iduronate-2-sulphatase. Purification, characterization and catalytic properties

Human iduronate-2-sulphatase (EC 3.1.6.13), which is involved in the lysosomal degradation of the glycosaminoglycans heparan sulphate and dermatan sulphate, was purified more than 500,000-fold in 5% yield from liver with a six-step column procedure, which consisted of a concanavalin A-Sepharose-Blue A-agarose coupled step, chromatofocusing, gel filtration on TSK HW 50S-Fractogel, hydrophobic separation on phenyl-Sepharose CL-4B and size separation on TSK G3000SW Ultrapac. Two major forms were identified. Form A and form B, with pI values of 4.5 and less than 4.0 respectively, separated at the chromatofocusing step in approximately equal amounts of recovered enzyme activity. By gel-filtration methods form A had a native molecular mass in the range 42-65 kDa. When analysed by SDS/PAGE, dithioerythritol-reduced and non-reduced form A and form B consistently contained polypeptides of molecular masses 42 kDa and 14 kDa. Iduronate-2-sulphatase was purified from human kidney, placenta and lung, and form A was shown to have similar native molecular mass and subunit components to those observed for liver enzyme. Both forms of liver iduronate-2-sulphatase were active towards a variety of substrates derived from heparin and dermatan sulphate. Kinetic parameters (Km and Kcat) of form A were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo, namely heparan sulphate, heparin and dermatan sulphate. Substrate with 6-sulphate esters on the aglycone residue adjacent to the iduronic acid 2-sulphate residue being attack were hydrolysed with catalytic efficiencies up to 200 times above that observed for the simplest disaccharide substrate without a 6-sulphated aglycone residue. The effect of incubation pH on enzyme activity towards the variety of substrates evaluated was complex and dependent on substrate aglycone structure, substrate concentration, buffer type and the presence of other proteins. Sulphate and phosphate ions and a number of substrate and product analogues were potent inhibitor of form A and form B enzyme activities.

Castanospermine-glucosides as selective disaccharidase inhibitors

Castanospermine (CS) is a potent but non-selective inhibitor of many glycohydrolases including the intestinal disaccharidases. Several CS-glucosides were synthesized to investigate the effect of an attached glucopyranosyl residue on the potency and selectivity of CS toward inhibition of intestinal disaccharidases. 8α-glucosyl-CS and 7α-glucosyl-CS were nearly as potent against sucrase activity as CS ( ic 50 values = 30, 40, and 20 nM respectively) but were 1/50 or less as potent as CS against lactase and trehalase activities. 8β-glucosyl-CS was 1/20 to 1/140 as potent as CS and 1α-glucosyl-CS was 1/57 to 1/1500 as potent as CS against disaccharidase activities. 1α-glc-CS was less selective than CS, whereas the other CS-glucosides were more selective. 7α-glc-CS and 8αḡlc-CS were the most sucrase selective and were particularly ineffective against trehalase and lactase activities. 8β-glc-CS was similar to CS except for relatively weaker trehalase inhibition. In summary, selectivity toward certain disaccharidases was achieved by glucosylation of CS hydroxyls. However, a simple structural comparison of the CS-glucoside to a disaccharide substrate did not reliably predict which disaccharidase would be more inhibited by the CS-glucoside.

Conversion of disaccharides to 3-ketodisaccharides by nongrowing and immobilized cells ofAgrobacterium tumefaciens

High-performance liquid chromatography was used to estimate 3-ketolactose and 3-ketosucrose in cultures of agrobacteria. The activities of enzymes that convert the disaccharide substrate were evaluated during batch cultivation ofAgrobacterium tumefaciens on sucrose, maltose, and lactose. The highest activity of glucoside 3-dehydrogenase and a slight activity of disaccharide-hydrolyzing enzymes were found in cells grown on lactose. Nongrowing cells converted lactose to 3-ketolactose faster than immobilized cells did. Immobilization of cells into polysaccharide gels did not stabilize the activity of glucoside 3-dehydrogenase. Glutaraldehyde cross-linking of the cell content led to an inactivation of the respiratory chain but Fe3+ could be used as an electron acceptor. Cells treated with glutaraldehyde converted lactose faster than nongrowing ones but the activity of glucoside 3-dehydrogenase was not stable.

Immunopurification and characterization of human α-l-iduronidase with the use of monoclonal antibodies

alpha-L-Iduronidase from human liver was purified by a three-step five-column procedure and by immunoaffinity chromatography with a monoclonal antibody raised against purified enzyme. Seven bands identified by staining with Coomassie Blue had molecular masses of 74, 65, 60, 49, 44, 18 and 13 kDa and were present in both preparations of the liver enzyme. However, relative to the immunopurification procedure, alpha-L-iduronidase purified by the five-column procedure was considerably enriched in the 65 kDa polypeptide band. The seven bands were identified by Western-blot analysis with two different monoclonal antibodies raised against alpha-L-iduronidase. The chromatographic behaviour of alpha-L-iduronidase on the antibody column was dependent upon the quantity of enzyme loaded. Above a particular load concentration a single peak of enzyme activity was eluted, whereas at load concentrations below the critical value alpha-L-iduronidase was eluted in two peaks of activity, designated form I (eluted first) and form II (eluted second). The following properties of the two forms of alpha-L-iduronidase were determined. (1) The two forms from liver were composed of different proportions of the same seven polypeptides. (2) When individually rechromatographed on the antibody column, each form from liver shifted to a more retarded elution position but essentially retained its chromatographic behaviour relative to the other form. (3) Forms I and II of liver alpha-L-iduronidase showed no difference in their activities towards disaccharide substrates derived from two glycosaminoglycan sources, heparan sulphate and dermatan sulphate. (4) The native molecular size of forms I and II of liver alpha-L-iduronidase was 65 kDa as determined by gel-permeation chromatography. (5) Immunoaffinity chromatography of extracts of human lung and kidney resulted in the separation of alpha-L-iduronidase into two forms, each with different proportions of the seven common polypeptide species. (6) Lung forms I and II were taken up readily into cultured skin fibroblasts taken from a patient with alpha-L-iduronidase deficiency. Liver forms I and II were not taken up to any significant extent. Lung form II gave intracellular contents of alpha-L-iduronidase that were more than double those of normal control fibroblasts, whereas lung form I gave contents approximately equal to normal control values. We propose that all seven polypeptides are derived from a single alpha-L-iduronidase gene product, and that different proportions of these polypeptides can function as a single alpha-L-iduronidase entity.(ABSTRACT TRUNCATED AT 400 WORDS)

Human liver glucuronate 2-sulphatase. Purification, characterization and catalytic properties

Human glucuronate 2-sulphatase (GAS), which is involved in the degradation of the glycosaminoglycans heparan sulphate and chondroitin 6-sulphate, was purified almost 2,000,000-fold to homogeneity in 8% yield from liver with a four-step six-column procedure, which consists of a concanavalin A-Sepharose/Blue A-agarose coupled step, a DEAE-Sephacel/octyl-Sepharose coupled step, CM-Sepharose chromatography and gel-permeation chromatography. Although more than 90% of GAS activity had a pI of greater than 7.5, other forms with pI values of 5.8, 5.3, 4.7 and less than 4.0 were also present. The pI greater than 7.5 form of GAS had a native molecular mass of 63 kDa. SDS/polyacrylamide-gel-electrophoretic analysis resulted in two polypeptide subunits of molecular mass 47 and 19.5 kDa. GAS was active towards disaccharide substrates derived from heparin [O-(beta-glucuronic acid 2-sulphate)-(1----4)-O-(2,5)-anhydro[1-3H]mannitol 6-sulphate (GSMS)] and chondroitin 6-sulphate [O-(beta-glucuronic acid 2-sulphate-(1----3)-O-(2,5)-anhydro[1-3H]talitol 6-sulphate (GSTS)]. GAS activity towards GSMS and GSTS was at pH optima of 3.2 and 3.0 respectively with apparent Km values of 0.3 and 0.6 microM respectively and corresponding Vmax values of 12.8 and 13.7 mumol/min per mg of protein respectively. Sulphate and phosphate ions are potent inhibitors of enzyme activity. Cu2+ ions stimulated, whereas EDTA inhibited enzyme activity. It was concluded that GAS is required together with a series of other exoenzyme activities in the lysosomal degradation of glycosaminoglycans containing glucuronic acid 2-sulphate residues.

Sanfilippo D syndrome: Estimation of N-acetylglucosamine-6-sulfatase activity with a radiolabeled monosulfated disaccharide substrate

N-Acetylglucosamine-6-sulfatase activity was assayed by incubation of the radiolabeled disaccharide O-( a-N-acetylglucosamine-6-sulfate)-(1 → 3)- l-[6- 3H]-idonic acid (GlcNAc6S-IdOA), with homogenates of leucocytes, cultured fibroblasts, and urine from normal individuals, patients affected with N-acetylglucosamine-6-sulfatase-deficiency (Sanfilippo D syndrome, mucopolysaccharidosis type IIID), and patients affected with other mucopolysaccharidoses and lysosomal storage disorders. The assay clearly distinguished affected homozygotes from their obligate heterozygotes and normal controls and other lysosomal storage disorders. Sulfatase activity in fibroblasts, leucocytes, and urine toward GlcNAc6S-IdOA exhibited a pH optimum at 4.2, 4.5, and 5.1, respectively. Sulfatase activity in fibroblasts had an apparent K m of 7.2 μ m and was significantly inhibited by both sulfate and phosphate ions. The action of fibroblast or leucocyte N-acetylglucosamine-6-sulfatase activity toward GlcNAc6S-IdOA is recommended for the routine enzymatic detection and classification of mucopolysaccharidosis type IIID patients.

Stability and substrate specificity of a β-glucosidase from the thermophilic bacterium Tp8 cloned into Escherichia coli

A facile isolation of β-glucosidase (EC 3.2.1.21) from Escherichia coli containing the recombinant plasmid pNZ1001 carrying a β-glucosidase gene from the extremely thermophilic anaerobic bacterium Tp8 is reported. The enzyme was purified to homogeneity by anion-exchange chromatography and steric exclusion HPLC following thermal denaturation/precipitation of heat-labile E. coli proteins. The enzyme had a broad specificity for β- d-glucosides, galactosides, fucosides, and xylosides. Action on aryl-β- d-glycosides of glucose, galactose, and fucose was characterized by low K m and high k cat K m values compared with disaccharide substrates for which specificity decreased in the order laminaribiose, sophorose, cellobiose, β-gentiobiose, lactose. Galactono-1-4-lactone, glucono-1-5-lactone, and 1- O-methyl-β- d-glucose were competitive inhibitors with K i values of 1.6, 0.09, and 17.5 m m, respectively. The enzyme was remarkably stable to detergents, urea, and organic solvents. Thermostability was greatest at the pH activity optimum (pH 6.0–6.5) and half-life ( t 1 2 ) values were 11 min at 90 °C, 105 min at 85 °C, and 900 min at 80 °C. Activity was destabilized by Sr 2+, Co 2+, Ca 2+, Mg 2+, and Mn 2+, but t 1 2 increased in the presence of substrates or competitive inhibitors. Activation energy, E a, was 54.3 kJ · mol −1. A free thiol group(s) was required for full activity, this being rapidly lost in the presence of Hg 2+ or N-ethyl maleimide.

Human liver N-acetylglucosamine-6-sulphate sulphatase. Purification and characterization.

Human N-acetylglucosamine-6-sulphate sulphatase was purified at least 50,000-fold to homogeneity in 78% yield from liver with a simple three-step four-column procedure, which consists of a concanavalin A-Sepharose/Blue A-agarose coupled step, chromatofocusing and Cu2+-chelating Sepharose chromatography. In all, four forms were isolated and partially characterized. Forms A and B, both with a pI greater than 9.5 and representing 30% and 60% respectively of the recovered enzyme activity, were separated by hydroxyapatite chromatography of the enzyme preparation obtained from the Cu2+-chelating Sepharose step. Both forms A and B had native molecular masses of 75 kDa. When analysed by SDS/polyacrylamide-gel electrophoresis, form A consists of a single polypeptide of molecular mass 78 kDa, whereas form B contained 48 kDa and 32 kDa polypeptide subunits. Neither form A nor form B was taken up from the culture medium into cultured human skin fibroblasts. The two other forms (C and D), with pI values of 5.8 and 5.4 respectively, represented approx. 7% and 3% of the total recovered enzyme activity. The native molecular masses of forms C and D were 94 kDa and approx. 75 kDa respectively. Form C contained three polypeptides with molecular masses of 48, 45 and 32 kDa. N-Acetylglucosamine-6-sulphate sulphatase activity was measured with a radiolabelled disaccharide substrate derived from heparin. The development of this substrate enabled the isolation and characterization of N-acetylglucosamine-6-sulphate sulphatase to proceed efficiently. Forms A, B and C had pH optima of 5.0, Km values of 11.7, 14.2 and 11.1 microM respectively and Vmax. values of 105, 60 and 53 nmol/min per mg of protein respectively. The molecular basis of the multiple forms of this sulphatase is not known. It is postulated that the differences in structure and properties of the four enzyme forms are due to differences in the state of processing of a large subunit.

Isolation and characterization of a 1,4-beta-D-glucan glucohydrolase from the yeast, Torulopsis wickerhamii.

1,4-beta-D-Glucan glucohydrolase (exo-1,4-beta-D-glucosidase) (EC 3.2.1.74) was isolated from growth supernatants of Torulopsis wickerhamii and was subjected to hydrodynamic, optical (CD), and kinetic analysis after purification to homogeneity by ammonium sulfate precipitation, size exclusion chromatography, ion exchange chromatography, and isopycnic banding centrifugation in cesium chloride. The last step was found to separate the enzyme from strongly associating, high molecular weight polysaccharide. Enzyme homogeneity was established by isoelectric focusing, sodium dodecyl sulfate-gel electrophoresis, and analytical high performance size exclusion chromatography using dual detection. The native exo-1,4-beta-D-glucosidase was found to be a dimer of 151,000 +/- 21,100 daltons by high performance size exclusion chromatography and 143,600 +/- 1,800 daltons by sedimentation equilibrium. The enzyme has a 12% linked carbohydrate content (mostly mannose) and no essential metal ions. Hydrolysis of p-nitrophenyl-beta-D-glucopyranoside was found to be optimal at pH 4.25 and 50 degrees C. The enzyme was found to produce beta-D-glucose from cellodextrins (indicating retention of anomeric configuration during hydrolysis) and demonstrated depolymerization from the non-reducing polymer terminus. The enzyme followed competitive type inhibition with p-nitrophenyl-beta-D-glucopyranoside as substrate and demonstrated high values of Ki for D-glucose and D-cellobiose inhibition (190 and 230 mM, respectively). The exo-1,4-beta-D-glucosidase was found to hydrolyze cellotetraose more rapidly than D-cellobiose and aryl-beta-D-glycosides more rapidly than all other substrates. Low levels of activity were found for the polymeric substrates beta-glucan (yeast cell walls), Avicel, and Walseth cellulose. Although this enzyme demonstrates broad disaccharide substrate specificity, a characteristic common to beta-D-glucosidases from many sources, the ability to hydrolyze higher cellodextrins more rapidly than cellobiose renders this enzyme the first exo-1,4-beta-D-glucosidase purified from yeast.