Human lysosomal alpha-glucosidase. Characterization of the catalytic site.

The substrate analogue conduritol B epoxide (CBE) is demonstrated to be an active site-directed inhibitor of human lysosomal alpha-glucosidase. A competitive mode of inhibition is obtained with glycogen as natural and 4-methylumbelliferyl-alpha-D-glucopyranoside as artificial substrate. The inactivation of the enzyme is time and concentration dependent and results in the covalent binding of CBE. Catalytic activity is required for binding to occur. CBE-labeled peptides containing the catalytic residue of lysosomal alpha-glucosidase were isolated and identified by microsequencing and amino acid analysis. The peptides appeared to originate from a protein domain which is highly conserved among alpha-amylases, maltase, glucoamylases, and transglucanosylases. Based on the sequence similarity and the mechanism of CBE binding, Asp-518 is predicted to be the essential carboxylate in the active site of lysosomal alpha-glucosidase. The functional importance of Asp-518 and other residues around the catalytic site was studied by expression of in vitro mutagenized alpha-glucosidase cDNA in transiently transfected COS cells. Substitution of Asp-513 by Glu-513 is shown to interfere with the posttranslational modification and the intracellular transport of the alpha-glucosidase precursor. The residues Trp-516 and Asp-518 are demonstrated to be critical for catalytic function.     

Overexpression, purification, and characterization of a barley alpha-glucosidase secreted by Pichia pastoris

alpha-Glucosidases (EC 3.2.1.20) are recognized as important in starch degradation during cereal seed germination. A barley (Hordeum vulgare) alpha-glucosidase expressed in Pichia pastoris was cultured in flasks; however, the yield was low necessitating the use of multiple batches. Problems arose because of significant variation between batches. We solved these problems by switching to a fermentation system producing a sufficient quantity of a uniform sample. Here we present the expression and purification of a recombinant alpha-glucosidase grown under fermentation conditions. We also present the results of experiments to characterize the thermostability, pH optimum, and substrate specificity of the recombinant enzyme. The optimal pH for the hydrolysis of maltose by recombinant alpha-glucosidase is between 3.5 and 4.5. The thermostability of recombinant alpha-glucosidase was determined at pH 4, where activity is optimal, and at pH 5 and 6, which better mimic the conditions used to convert barley starch to fermentable sugars during industrial processing. The results indicate the enzyme is most thermolabile at pH 4. However, the enzyme is protected from heat inactivation at pH 4 by high concentrations of sucrose. The purified enzyme hydrolyzed maltose three times more rapidly than nigerose and 20 times more rapidly than trehalose and isomaltose. Concentrations of maltose greater than 20 mM inhibited maltose hydrolysis. This is the first report of substrate inhibition for any alpha-glucosidase. The results indicate that the only significant difference between the recombinant enzyme and the previously characterized barley isoforms was the V(max) for maltose hydrolysis.     

Active site directed inhibition of a cytosolic beta-glucosidase from calf liver by bromoconduritol B epoxide and bromoconduritol F

Hydrolysis of p-nitrophenyl-beta-D-glucoside by cytosolic beta-glucosidase proceeds with retention of the anomeric configuration. Whereas inactivation of the enzyme by the glucosidase inhibitor conduritol B epoxide (CBE) was extremely slow (ki(max)/Ki 0.57 M-1 min-1) it reacted 130 times more rapidly with 6-bromo-6-deoxy-CBE (Br-CBE). The beta-glucosidase could be labeled with [3H]Br-CBE; incorporation of 1 mol inhibitor/mol enzyme resulted in complete loss of activity. Most of the bound inhibitor was released after denaturation and treatment with ammonia as (1,3,4/2,5,6)-6-bromocyclohexanepentol, thus demonstrating the formation of an ester bond with an active site carboxylate by trans-diaxial opening of the epoxide ring. It was concluded from the Ki values for the epoxide inhibitors and for coduritol B with the cytosolic enzyme and corresponding data for the lysosomal beta-glucosidase that the unusually low reactivity with CBE and Br-CBE is probably due to the inability of the cytosolic enzyme to effectively donate a proton to the epoxide oxygen. An extremely rapid inactivation of the cytosolic beta-glucosidase was caused by bromoconduritol F ((1,2,4/3)-1-bromo-2,3,4-trihydroxycyclohex-5-ene) with ki(max)/Ki 10(5) M-1 min-1. In contrast with the Br-CBE-inhibited enzyme the beta-glucosidase inhibited by bromoconduritol F was subject to spontaneous reactivation with t1/2 approximately 20 min.     

The maltase, glucoamylase and transglucosylase activities of acid α-glucosidase from rabbit muscle

1. The maltase and glucoamylase activities of acid alpha-glucosidase purified from rabbit muscle exhibited marked differences in certain physicochemical properties. These included pH stability, inactivation by thiol-group reagents, inhibition by alphaalpha-trehalose, methyl alpha-d-glucoside, sucrose, turanose, polyols, glucono-delta-lactone and monosaccharides, pH optimum and the kinetics and pH-dependence of cation activation. 2. The results are interpreted in terms of the existence of at least two specific substrate-binding sites or sub-sites. One site is specific for the binding of maltose and probably other oligosaccharides. The second site binds polysaccharides such as glycogen. 3. The sites appear to be in close proximity, since glycogen and maltose are mutually inhibitory substrates and interact directly in transglucosylation reactions. 4. Acid alpha-glucosidase exhibited intrinsic transglucosylase activity. The enzyme catalysed glucosyl-transfer reactions from [(14)C]maltose (donor substrate) to polysaccharides (glycogen and pullulan) and to maltose itself (disproportionation). The pH optimum was 5.1, with a shoulder or secondary activity peak at pH5.4. The glucose transferred to glycogen was attached by alpha-1,4- and alpha-1,6-linkages. Three major oligosaccharide products of enzyme action on maltose (disproportionation) were detected. 5. The kinetics of enzyme action on [(14)C]maltose showed that the rate of transglucosylation increased in a sigmoidal fashion as a function of substrate concentration, approximately in parallel with a decrease in the rate of glucose release. 6. The results are interpreted to imply competitive interaction at a specific binding site between maltose and water as glucosyl acceptors. 7. The results are discussed in terms of the possible existence of multiple subgroups of glycogen-storage disease type II.     

Effects of castanospermine on purified lysosomal alpha-1,4-glucosidase

The indolizidine alkaloid castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine) inhibits hydrolysis of maltose, glycogen and isomaltose by purified lysosomal alpha-glucosidase yielding Ki values of 0.095, 0.10 and 0.30 mumol/l, respectively. Castanospermine exhibited high affinity for both the maltose and isomaltose sites. In distinct contrast, the alkaloid exhibited little or no affinity for the site catalyzing hydrolysis of glycogen as indicated by a noncompetitive mode of inhibition. Kinetic data presented in this report indicate castanospermine to be a very potent inhibitor of lysosomal alpha-glucosidase.     

Alpha-glucosidase inhibitory activity of Syzygium cumini (Linn.) Skeels seed kernel in vitro and in Goto-Kakizaki (GK) rats

Syzygium cumini seed kernel extracts were evaluated for the inhibition of alpha-glucosidase from mammalian (rat intestine), bacterial (Bacillus stearothermophilus), and yeast (Saccharomyces cerevisiae, baker's yeast). In vitro studies using the mammalian alpha-glucosidase from rat intestine showed the extracts to be more effective in inhibiting maltase when compared to the acarbose control. Since acarbose is inactive against both the bacterial and the yeast enzymes, the extracts were compared to 1-deoxynojirimycin. We found all extracts to be more potent against alpha-glucosidase derived from B. stearothermophilus than that against the enzymes from either baker's yeast or rat intestine. In an in vivo study using Goto-Kakizaki (GK) rats, the acetone extract was found to be a potent inhibitor of alpha-glucosidase hydrolysis of maltose when compared to untreated control animals. Therefore, these results point to the inhibition of alpha-glucosidase as a possible mechanism by which this herb acts as an anti-diabetic agent.     

Intracellular activity of lysosomal glucosylceramidase measured with 4-nonylumbelliferyl beta-glucoside

Enzymatic activity of lysosomal glucosyl-ceramidase was determined in intact murine hybridoma and macrophage cells with the synthetic substrate nonylumbeliferyl-beta-glucoside (NUG). The substrate was applied as complex with bovine serum albumin (two binding sites, Kd 2.2 +/- 0.3 microM). The transport of the artificial substrate from medium to the enzyme was explored by measurements of substrate concentrations in cellular membranes and of endocytosis rate relative to substrate hydrolysis. The results indicated that, after enrichment in the plasma membrane, the substrate is mainly transported by simple diffusion. Release of nonylumberlliferone monitored fluorimetrically after disintegration of the cells in borate buffer containing Triton X-100 at pH 9.5 showed that 10(8) cells of both cell lines hydrolysed 1-1.5 nmol substrate/min at a total concentration of 0.1 mM NUG in the medium. Substrate hydrolysis was prevented by preincubating the cells with conduritol B epoxide (CBE), a specific active site-directed inhibitor of lysosomal glucosylceramidase. The substrate concentration at the site of the enzyme and maximal activity were evaluated by the inhibiting effect of the substrate on the inactivation rate by conduritol B epoxide. The rate of inhibitor uptake measured with bromo-[3H]conduritol B epoxide was shown to be not rate-limiting for the inactivation reaction. The molar concentration of the enzyme was determined by labeling with bromo-[3H]conduritol B epoxide. Comparison of the maximal intracellular activity with that of the enzyme after disintegration and activation by taurocholate showed a 20-fold lower activity in the native environment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel alpha-glucosidase from Aspergillus nidulans with strong transglycosylation activity

Aspergillus nidulans possessed an alpha-glucosidase with strong transglycosylation activity. The enzyme, designated alpha-glucosidase B (AgdB), was purified and characterized. AgdB was a heterodimeric protein comprising 74- and 55-kDa subunits and catalyzed hydrolysis of maltose along with formation of isomaltose and panose. Approximately 50% of maltose was converted to isomaltose, panose, and other minor transglycosylation products by AgdB, even at low maltose concentrations. The agdB gene was cloned and sequenced. The gene comprised 3,055 bp, interrupted by three short introns, and encoded a polypeptide of 955 amino acids. The deduced amino acid sequence contained the chemically determined N-terminal and internal amino acid sequences of the 74- and 55-kDa subunits. This implies that AgdB is synthesized as a single polypeptide precursor. AgdB showed low but overall sequence homology to alpha-glucosidases of glycosyl hydrolase family 31. However, AgdB was phylogenetically distinct from any other alpha-glucosidases. We propose here that AgdB is a novel alpha-glucosidase with unusually strong transglycosylation activity.     

A novel alpha-glucosidase from the moss Scopelophila cataractae

Scopelophila cataractae is a rare moss that grows on copper-containing soils. S. cataractae protonema was grown on basal MS medium containing copper. A starch-degrading activity was detected in homogenates of the protonema, after successive extraction with phosphate buffer and buffer containing 3 M LiCl. Buffer-soluble extract (BS) and LiCl-soluble extract (LS) readily hydrolyzed amylopectin to liberate only glucose, which shows that alpha-glucosidase (EC 3.2.1.20) in BS and LS hydrolyzed amylopectin. The K(m) value of BS for maltose was 0.427. The K(m) value of BS for malto-oligosaccharide decreased with an increase in the molecular mass of the substrate. The value for maltohexaose was 0.106, which is about four-fold lower than that for maltose. BS was divided into two fractions of alpha-glucosidase (BS-1 and BS-2) by isoelectric focusing. The isoelectric points of these two enzymes were determined to be 4.36 (BS-1) and 5.25 (BS-2) by analytical gel electrofocusing. The two enzymes readily hydrolyzed malto-oligosaccharides. The two enzymes also hydrolyzed amylose, amylopectin and soluble starch at a rate similar to that with maltose. The two enzymes readily hydrolyzed panose to liberate glucose and maltose (1 : 1), and the K(m) value of BS for panose was similar to that for maltotriose, whereas the enzymes hydrolyzed isomaltose only weakly. With regard to substrate specificity, the two enzymes in BS are novel alpha-glucosidases. The two enzymes also hydrolyzed beta-limit dextrin, which has many alpha-1,6-glucosidic linkages near the non-reducing ends, more strongly than maltose, which shows that they do not need a debranching enzyme for starch digestion. The starch-degrading activity of BS was not inhibited by p-chloromercuribenzoic acid or alpha-amylase inhibitor. When amylopectin was treated with BS and LS in phosphate buffer, pH 6.0, glucose, but not glucose-1-phosphate, was detected, showing that the extracts did not contain phosphorylase but did contain an alpha-glucosidase. These results show that alpha-glucosidases should be capable of complete starch digestion by themselves in cells of S. cataractae.     

Some properties of human liver acid alpha-glucosidase.

1. Albumin activates human liver acid alpha-glucosidase (alpha-D-glucoside hydrolase, EC 3.2.1.20). From the Arrhenius plot, pH-dependence and Lineweaver-Burk plots it can be concluded that this activation is not only due to stabilisation of the enzyme, but also influences the enzymatic activity. It is proposed that for optimal functioning human liver acid alpha-glucosidase needs a protein environment. 2. Glycogen has a competitive inhibitory effect on the hydrolysis of 4-methylumbelliferyl-alpha-D-glucopyranoside, in contrast to maltose which exhibits a non-competitive type of inhibition. It is concluded that two catalytic sites exist, one for glycogen and one for maltose, while both sites influence each other. With glycogen as substrate a break in the Arrhenius plot is found. This is not the case when maltose is used as substrate. 3. The effect of antibody raised against human liver acid alpha-glucosidase on the activity of human liver acid alpha-glucosidase is studied. No corss-reacting material could be demonstrated in the liver of a patient with glycogen storage disease Type II (M. Pompe, acid alpha-glucosidase deficiency).     

Maltose metabolism of Pseudomonas fluorescens.

Pseudomonas fluorescens W uses maltose exclusively by hydrolyzing it to glucose via an inducible alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20). No evidence for phosphorolytic cleavage or oxidation to maltobionic acid was found in this organism. The alpha-glucosidase was totally intracellular and was most active at pH of 7.0. Induction occurred when cells were incubated with maltotriose or maltose. Induction was rapid and easily detectable within the first 5 min after the addition of the inducer. Glucose and its derivatives did not repress induction. Cells growing on DL-alanine or succinate plus maltose exhibited lower levels of alpha-glucosidase than those grown on maltose alone or maltose plus glucose. Induction required both messenger ribonucleic acid and protein synthesis.     

Evidence for a single catalytic site of honeybee alpha-glucosidase I by chemical modification with diethylpyrocarbonate.

Honeybee alpha-glucosidase I was inactivated with diethylpyrocarbonate (DEPC). The inactivation followed pseudo-first-order kinetics. The rate of the loss of activity was decreased by the addition of a substrate, maltose. Since there was no spectral change in the tyrosine absorption region, it was recognized that DEPC did not react with this residue. The alpha-glucosidase had one free sulfhydryl group, which was not involved in the catalytic reaction, and was not modified by DEPC. On the other hand, the specific reaction of DEPC with a histidyl residue was spectrophotometrically confirmed by an increase in absorption near 240 nm, and the activity of the inactivated enzyme was restored by hydroxylamine. The modification rate of one histidyl residue by DEPC was almost equal to the rate of the activity loss. These results indicate that there is one histidyl residue at or near the catalytic site, and that honeybee alpha-glucosidase I has a single active site.     

Characterization of a neopullulanase and an alpha-glucosidase from Bacteroides thetaiotaomicron 95-1.

Previously, we constructed a gene disruption in the pullulanase I gene of Bacteroides thetaiotaomicron 5482A. This mutant, designated B. thetaiotaomicron 95-1, had a lower level of pullulanase specific activity than did wild-type B. thetaiotaomicron but still exhibited a substantial amount of pullulanase activity. Characterization of the remaining pullulanase activity present in B. thetaiotaomicron 95-1 has identified an alpha(1----4)-D-glucosidic bond cleaving pullulanase which has been tentatively designated a neopullulanase. The neopullulanase (pullulanase II) is a 70-kDa soluble protein which cleaves alpha(1----4)-D-glucosidic bonds in pullulan to produce panose. The neopullulanase also cleaved alpha(1----4) bonds in amylose and in oligosaccharides of maltotriose through maltoheptaose in chain length. An alpha-glucosidase from B. thetaiotaomicron 95-1 was characterized. The alpha-glucosidase was partially purified to a preparation containing three proteins of 80, 57, and 50 kDa. Pullulan and amylose were not hydrolyzed by the alpha-glucosidase. alpha(1----4)-D-Glucosidic oligosaccharides from maltose to maltoheptaose were hydrolyzed to glucose by the alpha-glucosidase. The alpha-glucosidase also hydrolyzed alpha(1----6)-linked oligosaccharides such as panose (the product of the pullulanase II action on pullulan) and isomaltotriose.     

Comparative study of glycosidase from cattle liver and exoglycanase from Aspergillus awamori]

Anomerities of products were estimated for glucosidases from cattle liver and Aspergillus awamori. It was demonstrated that the enzyme from cattle liver is alpha-glucosidase and that from Asp. awamori is exogluconase. It was demonstrated that alpha-glucosidase hydrolyzes the C1--O bond in the course of reaction. delta-Lactone of gluconic acid is a competitive inhibitor for both enzymes. The secondary kinetic isotope effects for both enzymes were measured. The isotope effect for alpha-glucosidase is equal to 1, for exogluconase 1,1 for glycogen and 1,18 for maltose. Some aspects of mechanisms of both enzymes are discussed in terms of the data obtained.     

Role of starch as a substrate for Bacteroides vulgatus growing in the human colon.

Bacteroides vulgatus is the numerically predominant Bacteroides species in the human colonic microflora. Unlike other colonic Bacteroides species, B. vulgatus is not a versatile utilizer of polysaccharides. The only types of polysaccharide that support rapid growth and high growth yields by all strains are the starches amylose and amylopectin. Amylase and alpha-glucosidase activities are among the highest found in a bacterial fraction obtained from human feces. This observation raised the question of whether B. vulgatus was the source of the fecal enzymes. Both alpha-glucosidase and amylase were produced at 20- to 40-fold-higher levels when B. vulgatus was grown on maltose, amylose, or amylopectin than when B. vulgatus was grown on glucose or other monosaccharides. Both enzymes had the same pI (4.6 to 5.0) and undenatured molecular weight (150,000). The pIs and molecular weights of the B. vulgatus amylase and alpha-glucosidase were the same as those of the fecal enzymes. To determine whether the B. vulgatus alpha-glucosidase was identical to the fecal alpha-glucosidase, we partially purified the B. vulgatus enzyme and raised an antiserum against it. Using this antiserum, we showed that all strains of B. vulgatus produced the same enzyme. The antiserum did not detect the B. vulgatus alpha-glucosidase in the bacterial fraction from human feces, even when a partially purified preparation of the fecal enzyme was used. Thus the alpha-glucosidase activity in the bacterial fraction from human feces is not the B. vulgatus enzyme.     

Role of starch as a substrate for Bacteroides vulgatus growing in the human colon

Bacteroides vulgatus is the numerically predominant Bacteroides species in the human colonic microflora. Unlike other colonic Bacteroides species, B. vulgatus is not a versatile utilizer of polysaccharides. The only types of polysaccharide that support rapid growth and high growth yields by all strains are the starches amylose and amylopectin. Amylase and alpha-glucosidase activities are among the highest found in a bacterial fraction obtained from human feces. This observation raised the question of whether B. vulgatus was the source of the fecal enzymes. Both alpha-glucosidase and amylase were produced at 20- to 40-fold-higher levels when B. vulgatus was grown on maltose, amylose, or amylopectin than when B. vulgatus was grown on glucose or other monosaccharides. Both enzymes had the same pI (4.6 to 5.0) and undenatured molecular weight (150,000). The pIs and molecular weights of the B. vulgatus amylase and alpha-glucosidase were the same as those of the fecal enzymes. To determine whether the B. vulgatus alpha-glucosidase was identical to the fecal alpha-glucosidase, we partially purified the B. vulgatus enzyme and raised an antiserum against it. Using this antiserum, we showed that all strains of B. vulgatus produced the same enzyme. The antiserum did not detect the B. vulgatus alpha-glucosidase in the bacterial fraction from human feces, even when a partially purified preparation of the fecal enzyme was used. Thus the alpha-glucosidase activity in the bacterial fraction from human feces is not the B. vulgatus enzyme.     

A thermostable acid alpha-glucosidase from Tetrahymena thermophila: purification and characterization

An acid alpha-glucosidase (EC 3.2.1.20) was purified to homogeneity from the culture medium of Tetrahymena thermophila CU 399. Its general molecular, catalytic and immunological properties were compared to those of the T. pyriformis W enzyme. The enzyme from T. thermophila was a 105-kD monomer and the N-terminus (25 amino acid residues) displayed some homology with that of T. pyriformis enzyme. The purified enzyme was most active at 56 degrees C and showed resistance to thermal inactivation. The acid alpha-glucosidase appears to have alpha-1,6-glucosidase as well as alpha-1,4-glucosidase activity. The Km values determined with p-nitrophenyl-alpha-glucopyranoside, maltose, isomaltose and glycogen were 0.7 mM, 2.5 mM, 28.5 mM and 18.5 mg/ml, respectively. The enzyme was antigenically distinct from T. pyriformis acid alpha-glucosidase.     

Purification and characterization of an alpha-glucosidase from a hyperthermophilic archaebacterium, Pyrococcus furiosus, exhibiting a temperature optimum of 105 to 115 degrees C.

Pyrococcus furiosus is a strictly anaerobic hyperthermophilic archaebacterium with an optimal growth temperature of about 100 degrees C. When this organism was grown in the presence of certain complex carbohydrates, the production of several amylolytic enzymes was noted. These enzymes included an alpha-glucosidase that was located in the cell cytoplasm. This alpha-glucosidase has been purified 310-fold and corresponded to a protein band of 125 kilodaltons as resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme exhibited optimum activity at pH 5.0 to 6.0 and over a temperature range of 105 to 115 degrees C. Kinetic analysis conducted at 108 degrees C revealed hydrolysis of the substrates p-nitrophenyl-alpha-D-glucopyranoside (PNPG), methyl-alpha-D-glucopyranoside, maltose, and isomaltose. Trace activity was detected towards p-nitrophenyl-beta-D-glucopyranoside, and no activity could be detected towards starch or sucrose. Inhibition studies conducted at 108 degrees C with PNPG as the substrate and maltose as the inhibitor yielded a Ki for maltose of 14.3 mM. Preincubation for 30 min at 98 degrees C in 100 mM dithiothreitol and 1.0 M urea had little effect on enzyme activity, whereas preincubation in 1.0% sodium dodecyl sulfate and 1.0 M guanidine hydrochloride resulted in significant loss of enzyme activity. Purified alpha-glucosidase from P. furiosus exhibited remarkable thermostability; incubation of the enzyme at 98 degrees C resulted in a half life of nearly 48 h.     

Hepatic microsomal epoxide hydrase. Involvement of a histidine at the active site suggests a nucleophilic mechanism

The effects of a wide variety of chemical modification reagents on the activity of purified rat liver microsomal epoxide hydrase have been investigated. Alkylating agents, such as the phenacyl bromides and benzyl bromide are potent inhibitors of epoxide hydrase. 2-Bromo-4'-nitroacetophenone (p-nitrophenacyl bromide) specifically and irreversibly inactivates epoxide hydrase. Pseudo-first order kinetics of inhibition is observed at higher inhibitor/enzyme ratios. The rate of inactivation is controlled by a group on the enzyme with an apparent pKa of 7.6. Inactivation of the enzyme with 14C-labeled 2-bromo-4'-nitroacetophenone leads to the incorporation of approximately 1 mol of radioactive inhibitor/mol of protein. Epoxide hydrase can be protected against this inactivation by the substrate phenanthrene-9,10-oxide. These results are consistent with the interpretation that 2-bromo-4'-nitroacetophenone acts as an active site-directed inhibitor. The site of alkylation by 2-bromo-4'-nitroacetophenone is a histidine residue of epoxide hydrase. The N-alkylated histidine derivative has been identified as 1-(p-nitrophenacyl)-4-histidine. A possible mechanism for the enzymatic hydration catalyzed by epoxide hydrase is discussed which involves a histidine residue of the enzyme serving as a general base catalyst for the nucleophilic addition of water.