Porcine Pancreatic Alpha-amylase Research Articles

Differential chemical modification of substrate binding areas in porcine-pancreatic alpha-amylase by three regioisomeric photolabile ligands

Three regioisomeric radiolabelled spacer-modified oligosaccharides: methyl 4′- O-(4- S-(3-azi-4-α- d-glucopyranosyloxy-1-[ 3H]butyl)-6-deoxy-4-thio-α- d- xylo-hex-5-enopyranosyl]-α-maltoside ( 12a, G1-G3*), methyl 4- O-[4- S-(3-azi-4-α-maltosyloxy-1-[ 3H]butyl)-6-deoxy-4-thio-α- d- xylo-hex-5-enopyranosyl]-α- d-glucopyranoside ( 15a, G2-G2*) and methyl 4- S-(3-azi-4-α-maltotriosyloxy-1-[ 3H]butyl)-6-deoxy-4-thio-α- d- xylo-hex-5-enopyranoside ( 16a, G3-G1*) were synthesised and used as photoaffinity probes for the chemical modification of porcine-pancreatic alpha-amylase (PPA). Incorporation of covalently attached radioactivity amounted to 25–38% of the stoichiometric value. Tryptic digestion of the three labelled protein preparations PPA-G1-G3*, PPA-G2-G2*, and PPA-G3-G1* and the purification of the labelled peptides by fractional HPLC yielded altogether six pure components. On the basis of the published three-dimensional structure peptides G1-G3-II, G2-G2-II, and G2-G2-III were part of the catalytic site. G1-G3-I and G2-G2-I were part of the surface binding site. The major component derived from PPA, labelled by G3-G1*, corresponded to an area that is neither close to the active site nor to the surface starch-binding domain, which clearly indicates the presence of a third, hitherto undetected, substrate-binding site.

Enzymatic method for assaying calcium in serum and urine with porcine pancreatic alpha-amylase

We developed a kinetic assay for calcium in serum and urine, based on the activation of porcine pancreatic alpha-amylase (EC 3.2.1.1) with 2-chloro-4-nitrophenyl-alpha-maltotrioside as substrate. The kinetic generation of 2-chloro-4-nitrophenol, monitored at 405 nm, was proportional to the concentration of calcium in serum and urine. The assay was developed and evaluated with the Cobas Bio centrifugal analyzer. The average within-run and between-day imprecision (CV) was 0.96/1.26% and 1.07/1.63%, respectively, for serum calcium and 1.50/2.54% and 1.70/2.64%, respectively, for urine calcium. Results of the proposed method (y) correlated well with those determined by atomic absorption spectrophotometry (x): y(serum) = 1.005x + 0.028 mmol/L (Sy/x = 0.058, r = 0.974, n = 50), and y(urine) = 1.017x-0.115 mmol/L Sy/x = 0.30, r = 0.981, n = 25). We also present data showing that the method is highly sensitive, rapid, relatively free of interference, and amenable to automation.

Effect of modifying histidine residues on the action of Bacillus amyloliquefaciens and barley-malt α-amylases

Modification of porcine pancreatic alpha-amylase (PPA) and Taka-amylase A(TAA) with diethyl pyrocarbonate (DEP) causes activation of the release of p-nitrophenol from p-nitrophenol alpha-maltoside (G2PNP), and a decrease in amylase activity (hydrolysis of alpha-1,4 glucosidic bonds in starch). Among the possible sites of modification, attention focuses on three histidine residues present around the active site of alpha-amylases of many different origins. In PPA these are His 101, His 201, and His 299, with His 101 and His 299 being very close to the site of catalysis and thus perhaps directly or indirectly involved in the catalysis. On the other hand, His 201 is located on the aglycon side of the catalytic site, and we have suggested that it is involved in the increase of PNP release after chemical modification. Investigations of site-directed mutagenesis of the histidine residues of human pancreatic alpha-amylase support this identification. Although the degree of sequence similarity among alpha-amylases of different origins is low, there are several conserved short regions. Most belong to the structural components of the active site in PPA and TAA. Furthermore, there is a close similarity in the three-dimensional structures of PPA and TAA. The conserved residues around the active site in all alpha-amylases suggest some universal structural similarities in these active sites. Therefore, we examined the effects of the chemical modification of histidine residues in Bacillus amyloliquefaciens alpha-amylase(BLA) with DEP and made a comparison with modification of barley alpha-amylase isoenzyme II(BAII), using identical substrate systems. These two alpha-amylases have more substrate binding subsites than PPA and TAA, and have similar action patterns with malto-oligosaccharides.

Effect of heat-moisture treatment on the structure and physicochemical properties of cereal, legume, and tuber starches

Native wheat, oat, lentil, yam, and potato starches were heat treated at 100°C for 16 h at moisture contents between 10 and 30%. The heat treatment did not change granule size and shape. In oat starch, granules were less compactly packed after heat treatment. The X-ray diffraction intensities increased in wheat, oat, and lentil starches, but decreased in potato and yam. The X-ray patterns of wheat and oat starches remained unchanged, while those of lentil, potato, and yam starches became more cereal-like. In all starches, the swelling factor and amylose leaching decreased, being more pronounced in potato. Heat treatment induced complex formation between amylose and native lipids. Differential scanning calorimetry of the heat-treated samples showed a broadening of the gelatinization-temperature range and a shifting of the endothermal transition towards higher temperatures. These changes were more pronounced in potato starch. The gelatinization enthalpy of wheat, oat, and lentil starches remained unchanged, but those of potato and yam starches decreased on heat treatment. Heat treatment increased the 95°C viscosity of wheat starch, but decreased those of oat, lentil, potato, and yam starches. In all starches, thermal and shear stability increased after heat treatment. Acid hydrolysis decreased on heat treatment of wheat and lentil starches, but increased in oat, potato, and yam starches. However, in potato and yam starches, the foregoing trend was evident only during the first seven days of hydrolysis. Thereafter, acid hydrolysis was more pronounced in native than in heat-treated starches. The susceptibility towards hydrolysis by porcine pancreatic alpha amylase decreased on heat treatment of wheat and lentil starches, whereas increases were observed for oat, potato, and yam starches. The results indicated that the extent of starch-chain associations within the amorphous regions and the degree of crystalline order are altered during heat-moisture treatment. The magnitude of these changes were found to be dependent upon the moisture content during heat treatment and on the starch source.

Studies on the physicochemical properties of native, defatted, and heat-moisture treated pigeon pea ( Cajanus cajan L) starch

Starch from pigeon pea ( Cajanus cajan L) was isolated and some of the important characteristics determined. The yield of startch was 29.7% on a whole seed basis. The shape of the starch granule was oval to eliptical to irregular, with granules 8–32 μm in diameter. Scanning electron micrographs revealed the presence of smooth surfaces with a large number of grooves. The gelatinization temperature-range was 347–354–360 K and the total amylose content was 29.3%, of which 2.7% was complexed by native lipids. The starch exhibited a restricted two-stage swelling pattern and a moderate solubility in water. The viscoamylographic examination of the starch paste (6% w/v) showed the absence of a peak viscosity, a low 95°C viscosity (80 Brabender units, BU) and an increase in consistency (140 BU) during the holding cycle at 95°. Native granules were very resistance to hydrolysis by porcine pancreatic alpha amylase and 2.2 M HCl. The X-ray diffraction pattern was of the C-type. The results showed that the starch chains in the amorphous regions of pigeon pea were more highly associated than those of other legume starches. Defatting (hot 3:1 1-propanol-water) and heat-moisture treatment (100°C, 16 h, 30% moisture) increased the gelatinization temperature and broadened the gelatinization temperature range, with relatively little change in gelatinization enthalpy. Defatting and heat-moistuer treatment also increased the pasting temperature and susceptibility towards alpha-amylase, but decreased the swelling factor, amylose leaching, and paste consistencies at 95 and 50°C. However, the relative crystallinity remained unchanged. Acid hydrolysis increased on defatting but decreased on heat-moisture treatment. Defatting caused greater changes to the granular surface than heat-moisture treatment.

Substrate recognition at the binding site in mammalian pancreatic .alpha.-amylases

Porcine and human pancreatic alpha-amylases (PPA and HPA, respectively) have five binding sites for hexose residues of substrates. As previously reported, when a substrate is too small to occupy subsite 5 of the alpha-amylases, the optimum pH for catalytic activity changes from neutral to acidic [Ishikawa, K., Matsui, I., Honda, K., & Nakatani, H. (1990) Biochemistry 29, 7119-7123]. We studied the mechanism by which the enzyme recognizes the substrate by using the synthetic substrates maltopentaose (G5) and its analogs 4-O-alpha-maltotetraosyl-D-xylose (G4-X), 4-O-alpha-maltotetraosyl-2-deoxy-D-glucose (G4-D), 3-O-alpha-maltotetraosyl-L- sorbose (G4-S), 4-O-beta-maltotetraosyl-D-glucose (G4 beta-G), alpha-maltotetraosyl-beta-D-fructose (G4-F), p-nitrophenyl alpha-maltotetraoside (G4-phi), and maltopentaitol (G4-GOH). The reducing-end residues of these substrates, relevant to subsite 5, are D-xylose (X), 2-deoxy-D-glucose (D), L-sorbose (S), D-glucose (G), alpha-D-fructofuranose (F), p-nitrophenyl (phi), and D-sorbitol (GOH), respectively. The optimum pH for catalytic activity on the substrates G5, G4-X, G4-D, G4-S, G4 beta-G, and G4-phi was neutral, while that for G4-GOH and G4-F was acidic. These results indicate that only six-membered-ring residues and a phenyl group are recognizable by subsite 5. The neutral pH profile for G4-phi suggests that steric compatibility of the substrates at subsite 5 is also important for the enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)

SUBUNIT STRUCTURES AND ESSENTIAL AMINO ACID RESIDUES OF WHITE KIDNEY BEAN (PHASEOLUS VULGARIS) ?-AMYLASE INHIBITORS

Both white kidney bean alpha-amylase inhibitors WKB 858A (MW 42,000) and WKB 858B (MW 20,000) were composed of two subunits as determined by N-terminal amino acid analysis; by amino acid sequence, by SDS-PAGE and by separation on a chromatofocusing column in 8 M urea. N-Terminal amino acids for Inhibitor WKB 858A were alanine and glycine, with a sequence of H2N-Ala-Glu-Asn-Ala- Gly-Thr-Tyr----COOH for deglycosylated 19,000 MW peptide and H2N-Gly-Asn ----COOH for deglycosylated 12,000 MW peptide. N-Terminal amino acids for Inhibitor WKB 858B were alanine and serine, with a sequence of H2N-Ala-Thr -Glu-Thr-Ser----COOH for the deglycosylated 9,000 MW peptide and H2N-Ser -Ala- Val-Gly-Leu-Asp-Phe-Val-Leu-Val-Pro-Val-Gln-Pro-Glu-Ser-Lys -Gly-Asp -Thr-Val-Thr-Val-Glu-Phe-Asp----COOH for the deglycosylated 15,000 MW peptide. Chemical modification of 2 of 7 His residues with diethylpyrocarbonate resulted in 26% loss of inhibitory activity. Modification of 1.5 of 7 Trp residues with N-bromosuccinimide gave 60% loss of inhibitory activity. Modification of 2 of 6 Tyr residues with N-acetylimidazole gave 60% loss of inhibitory activity. Modification of 3.6 of 6 Arg residues with p-hydroxyphenylglyoxal gave 64% loss of inhibitor activity. These results indicate the possible importance of one or more His, Trp, Tyr and perhaps Arg residues for inhibitory activity against porcine pancreatic alpha-amylase.

Strong competitive inhibition of porcine pancreaticalpha-amylase by aminodeoxy derivatives of maltose and maltotriose

The syntheses are described of 6-amino-6-deoxymaltose (2), the 6-amino-6-deoxy ( 4), 6′-amino-6′-deoxy ( 6), and 6″-amino-6″-deoxy ( 8) derivatives of maltotriose, and the methyl α- ( 10) and β-glycoside ( 12) and the 1-deoxy derivative ( 16) of 4. The Ki values (AM) of these competitive inhibitors of porcine pancreatic alpha-amylase were: 2, 88; 4, 1.9; 6, 2.0; 8, 175; 10, 360; 12, 9000; 16, 7600 (cf. 1800 for maltotriose and 3000 for methyl a-maltotrioside). The low values for 4 and 6 reflect reinforcement of the normal binding by ionic attraction and, possibly, interaction of the reducing end groups with the protein.

Mechanism of the adsorption of pancreatic alpha-amylase onto starch crystallites

The mechanism of the adsorption of alpha-amylase onto crystalline starch is still little understood ‘-’ despite its involvement in many industrial applications (degradation of starch/saccharides 6, purification of enzymes 3,4) and digestion phenomena. Nutritionally, the resistance of crystalline starch to alpha-amylase has received attention for both its hypoglycemic 7*8 and fibre-like effects 9-12. Adsorption onto solid starch has been observed for many alpha-amylases (bacterial 3-5,13, mammalian ‘*13) but examples of non-adsorption have been found (fungal i,13, bacterial ‘,13) The adsorption mechanism in terms 5 of molecular interactions has not yet been clarified. Studies of the adsorption of alpha-amylase are often complicated by numerous other parameters lv2. Thus, the porosity, adsorption surface, crystallinity, and susceptibility to hydrolysis of starch may vary significantly depending on the botanical source or the degree of damage i4. The aim of this work was to characterise the interactions of alpha-amylase and crystalline starch in a model system in which all of these parameters are controlled. Starch usually occurs as semi-crystalline structures containing interconnected amorphous and crystalline regions. The crystalline fraction consists of a three-dimensional aggregate of double helices formed from (1 + 4)-Lu-o-glucan segments. The .double helices adopt well-defined arrays (types A or B), the characteristics of which are now well known “sl’j. Ring et al. l7 by spherulitic recrystallisation from , concentrated solutions, obtained highly crystalline starch structures, the porosity and surface of which could be determined easily. Porcine pancreatic alpha-amylase (PPA), which occurs as the isoenzymes PPA-I and PPA-II, is the best known alpha-amylase ‘*,i9 and is responsible for the digestion of starchy materials. PPA-I is composed of three distinct domains

Site-directed mutagenesis of active site residues in Bacillus subtilis α-amylase

Site-directed mutagenesis of Bacillus subtilis N7 alpha-amylase has been performed to evaluate the roles of the active site residues in catalysis and to prepare an inactive catalytic-site mutant that can form a stable complex with natural substrates. Mutation of Asp-176, Glu-208, and Asp-269 to their amide forms resulted in over a 15,000-fold reduction of its specific activity, but all the mutants retained considerable substrate-binding abilities as estimated by gel electrophoresis in the presence of soluble starch. Conversion of His-180 to Asn resulted in a 20-fold reduction of kcat with a 5-fold increase in Km for a maltopentaose derivative. The relative affinities for acarbose vs. maltopentaose were also compared between the mutants and wild-type enzyme. The results are consistent with the roles previously proposed in Taka-amylase A and porcine pancreatic alpha-amylase based on their X-ray crystallographic analyses, although different pairs had been assigned as catalytic residues for each enzyme. Analysis of the residual activity of the catalytic-site mutants by gel electrophoresis has suggested that it derived from the wild-type enzyme contaminating the mutant preparations, which could be removed by use of an acarbose affinity column; thus, these mutants are completely devoid of activity. The affinity-purified mutant proteins should be useful for elucidating the complete picture of the interaction of this enzyme with starch.

Inhibition and binding modes of low-molecular-weight inhibitors of porcine pancreatic alpha-amylase.

Inhibition of porcine pancreatic alpha-amylase (1,4-alpha-D-glucan glucanohydrase) [EC 3.2.1.1] with maltotriitol (G3OH) and 4-phenylimidazole was investigated by using maltohexaitol (G6OH) and p-nitrophenyl-alpha-D-maltoside (G2PNP) as substrates. When G6OH was the substrate, both G3OH and 4-phenylimidazole behaved as competitive inhibitors. On the other hand, when G2PNP was the substrate, G3OH behaved as a competitive inhibitor, whereas 4-phenylimidazole behaved as a non-competitive inhibitor. Further inhibition study in the presence of both G3OH and 4-phenylimidazole, with G6OH as the substrate, showed that the two inhibitors compete with each other for the active site of the enzyme. Based on a consideration of the productive (reactive) binding modes of G2PNP and G6OH, and a nonproductive (nonreactive) binding mode of G2PNP, it is suggested that the binding sites of the two inhibitors may be partially overlapping around the catalytic site of the enzyme and that the rest of the binding site of each inhibitor lies along the substrate binding cleft of the enzyme.

Photolabile derivatives of maltose and maltotriose as ligands for the affinity labelling of the maltodextrin-binding site in porcine pancreatic alpha-amylase

The 3-azibutyl group was linked through sulfur to the anomeric position of maltose and maltotriose to yield the photolabile thioglycosides 3-azibutyl 1-thio-α-maltoside ( 11) and 3-azibutyl 1-thio-α-maltotrioside ( 12), and to the 4′- and 6′-position of maltose to give the thioethers 4′- S-(3-azibutyl)-4′-thiomaltose ( 8) and 6′- S-(3-azibutyl)-6′-thiomaltose ( 15). All four compounds were good competitive inhibitors of the action of porcine pancreatic alpha-amylase. Compound 12 irreversibly deactivated the enzyme to ∼ 100% when irradiated together with the protein. The other compounds were much less effective. It is likely that separate areas of the enzyme binding site are chemically modified by the different ligands.

Effect of limited proteolysis in the 8th loop of the barrel and of antibodies on porcine pancreas amylase activity

The porcine pancreatic alpha-amylase is a (beta/alpha)8-barrel protein, containing domains A and B (peptide sequence 1-403) and a distinct C-domain (peptide sequence 404-496). Separation of the terminal C-domain from the A and B domains has been attempted by limited proteolysis in the hinge region. Subtilisin was found to hydrolyse amylase between residues 369 and 370 situated in the loop between the eighth beta-strand and alpha-helix. The cleaved amylase was isolated by chromatofocusing and found to retain about 60% of the activity of the native enzyme, while the isolated fragments were inactive. Antigen binding fragments prepared from polyclonal antibodies to native amylase and the CNBr-fragment P1 (peptide sequence 395-496) respectively, were tested for influence on the enzyme activity. Antibodies directed against P1 had no effect whereas antibodies against the peptide sequence 1-394 and amylase respectively inhibited hydrolysis of substrates having four or more glucose residues but not of shorter oligomaltosides. Crystallographic analysis revealed that changes in the region of residue 369 might affect the conformation of the active site as well as of a second binding site. This site, located on the enzyme surface, is proposed to be required for the hydrolysis of larger substrates.

Detection of a covalent intermediate in the mechanism of action of porcine pancreatic alpha-amylase by using 13C nuclear magnetic resonance.

The catalytic mechanism of porcine pancreatic alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) has been examined by nuclear magnetic resonance (NMR) at subzero temperatures by using [1-13C]maltotetraose as substrate. Spectral summation and difference techniques revealed a broad resonance peak, whose chemical shift, relative signal intensity and time-course appearance corresponded to a beta-carboxyl-acetal ester covalent enzyme-glycosyl intermediate. This evidence supports a double-displacement covalent mechanism for porcine pancreatic alpha-amylase-catalyzed hydrolysis of glycosidic linkages, based on the presence of catalytic aspartic acid residues within the active site of this enzyme.

Three dimensional structure of porcine pancreatic alpha-amylase at 2.9 A resolution. Role of calcium in structure and activity.

The crystal structure of porcine pancreatic alpha-amylase (PPA) has been solved at 2.9 A resolution by X-ray crystallographic methods. The enzyme contains three domains. The larger, in the N-terminal part, consists of 330 amino acid residues. This central domain has the typical parallel-stranded alpha-beta barrel structure (alpha beta)8, already found in a number of other enzymes like triose phosphate isomerase and pyruvate kinase. The C-terminal domain forms a distinct globular unit where the chain folds into an eight-stranded antiparallel beta-barrel. The third domain lies between a beta-strand and a alpha-helix of the central domain, in a position similar to those found for domain B in triose phosphate isomerase and pyruvate kinase. It is essentially composed of antiparallel beta-sheets. The active site is located in a cleft within the N-terminal central domain, at the carboxy-end of the beta-strands of the (alpha beta)8 barrel. Binding of various substrate analogues to the enzyme suggests that the amino acid residues involved in the catalytic reaction are a pair of aspartic acids. A number of other residues surround the substrate and seem to participate in its binding via hydrogen bonds and hydrophobic interactions. The 'essential' calcium ion has been located near the active site region and between two domains, each of them providing two calcium ligands. On the basis of sequence comparisons this calcium binding site is suggested to be a common structural feature of all alpha-amylases. It represents a new type of calcium-protein interaction pattern.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics of enzymic hydrolysis of malto-oligosaccharides: A comparison with acid hydrolysis

The hydrolysis of malto-oligosaccharides G3-G6 catalysed by porcine pancreatic alpha-amylase was investigated kinetically at 25°. Kinetic parameters corresponding to different positions of enzymic attack were determined and product inhibition was evaluated. The enzymic hydrolysis was compared in terms of reaction rate and pattern of action with hydrolysis in 0.1m H2SO4 at 70°. Mathematical models for the mechanism of hydrolysis were developed and a good rationalisation of the experimental results was achieved.

An alpha-amylase inhibitor from cranberry bean (Phaseolus vulgaris): its specificity in inhibition of mammalian pancreatic alpha-amylases and formation of a complex with the porcine enzyme.

A proteinaceous inhibitor that inhibits mammalian alpha-amylases was prepared from cranberry bean and examined for its reactivity with alpha-amylases from various origins. The cranberry bean alpha-amylase inhibitor (CBAI) exhibited inhibitory effects on pancreatic alpha-amylases from the following mammals: pig, dog, cat, horse, sheep, cow, rabbit, guinea pig, rat, and mouse. CBAI showed a maximal inhibition at pH 5.5 against porcine pancreatic alpha-amylase (PPA). It was confirmed by gel filtration that a complex was formed in the 1:1 ratio between CBAI and PPA when they were incubated at 37 degrees C for 30 min at pH 5.5. A similar inhibition pattern was also observed at pH 6.9 that is optimal for the amylase reaction, but much higher concentrations of CABI were required to give 50% inhibition at pH 6.9 than at pH 5.5. Especially, both bovine and rat alpha-amylases were virtually unreactive to CBAI at pH 6.9.

Actions of Aspergillus oryzae alpha-amylase, potato phosphorylase, and rabbit muscle phosphorylase a and b on phosphorylated (1→4)-α- d-glucan

Aspergillus oryzae alpha-amylase [(1→4-α- d-glucan glucanohydrolase, EC 3.2.1.1] produced O-6-phosphoryl-α- d-glucopyranosyl)-(1→4)- O-α- d-glucopyranosyl-(1→4)- d-glucopyranose (6 3-phosphorylmaltotriose) and O-α- d-glucopyranosyl-(1→4)- O-(3-phosphoryl-α- d-glucopyranosyl)-(1→4)- O-α- d-glucopyranosyl-(1→4)- d- glucopyranose (3 3-phosphorylmaltotetraose) from potato starch upon exhaustive hydrolysis. These products indicate that the enzyme hydrolyses the same linkages in the vicinity of the 6-phosphorylated residue as porcine-pancreatic alpha-amylase, but hydrolyses different linkages in the vicinity of the 3-phosphorylated residue when compared with B. subtilis and pancreatic alpha-amylases. Potato phosphorylase [(1→4)-α- d-glucan:orthophosphate α- d-glucosyl-transferase, EC 2.4.1.1] and rabbit muscle phosphorylase a and b were unable to by-pass the phosphorylated d-glucosyl residue of 6-phosphorylated (1→4)-α- d-glucan, leaving three d-glucosyl residues attached to the 6-phosphorylated residue on the non-reducing side.

Separation and Characterization of Four Different Amylases ofEntamoeba histolytica.II. Characterization of Amylases

Purified E. histolytica amylases III to VI were characterized by their hydrolytic behaviour towards 4-nitrophenyl alpha-malto-oligosaccharides, malto-oligosaccharides, amylose, amylopectin, glycogen and Y-cyclodextrin. The influence of specific inhibitors on the amylase activity of E. histolytica was examined and compared with typical alpha- and beta-amylases. Amylases III and IV showed alpha-glucosidase and glucosyltransferase activity by cleaving terminal non-reducing glucose from pNPG1 (III, IV) and pNPG2 to pNPG7 (III). Both enzymes were able to cleave malto-oligosaccharides and glucopolysaccharides to a large number of malto-oligosaccharides. Also transglucosidation reactions were observed, but maltose was not hydrolysed. Amylase V showed exoamylase-like properties by preferentially cleaving maltose units from the non-reducing end of synthetic and biogenic malto-oligosaccharides by a multiple-attack mechanism. Amylase VI was characterized as an alpha-amylase, showing great similarities with porcine pancreatic alpha-amylase in the hydrolysis pattern of 4-nitrophenyl alpha-malto-oligosaccharides and glucopolysaccharides. With biogenic malto-oligosaccharides amylase VI showed a transglucosidation reaction.

Porcine-pancreatic alpha amylase hydrolysis of substrates containing 6-deoxy- d-glucose and 6-deoxy-6-fluoro- d-glucose and the specificity of subsite binding

Hydrolysis of 6-deoxyamylose and mono-6-deoxy-6-fluorocyclomaltoheptaose by porcine-pancreatic alpha amylase produces low-molecular-weight modified products, which have been analyzed by chemical and chromatographic techniques. Results for both substrates show that modified d-glucose and two isomers of modified maltoses are produced in the enzyme reaction. In addition, the formation of maltoses modified in the nonreducing residue is more favored than the formation of maltoses modified in the reducing residue. These results indicate that productive binding of 6-fluoro- and 6-deoxy- d-glucose residues is permitted at subsites 1 through 4 of the amylase-active site but that binding of these modified residues may be less favorable at subsite 3, the subsite at which catalytic attack occurs.