Difference in transglycosylation between human pancreatic and salivary alpha-amylases

Transglycosylation reactions of alpha-amylases from human pancreatic juice and saliva were examined by using O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose as a substrate and O-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-1-deoxy-1-[(2-pyridyl)amino]-D-glucitol as an acceptor. The transfer reaction was estimated by quantitation of O-alpha-D-glucopyranosyl-(1 leads to 4)-1-deoxy-1-[(2-pyridyl)amino]-D-glucitol produced by the enzymes from the transfer products, because the acceptor was not hydrolyzed. The amount of O-alpha-D-glucopyranosyl-(1 leads to 4)-1-deoxy-1-[(2-pyridyl)amino]-D-glucitol in the digest with pancreatic alpha-amylase was six times that in the digest with salivary alpha-amylase at the stage when the substrate was completely consumed, and the difference increased gradually on further incubation. The phenomenon can be applied to differentiate the two alpha-amylases in human serum.     

Preparation of non-reducing-end substituted p-nitrophenyl alpha-maltopentaoside (FG5P) as a substrate for a coupled enzymatic assay for alpha-amylases

p-Nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha-D - glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside, FG5P, was prepared, taking advantage of the action of Bacillus macerans cyclodextrin glucanotransferase on a mixture of O-6-deoxy-6-[(2-pyridyl)-amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha- D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-D-glucose and p-nitrophenyl alpha-glucoside. The maltopentaose derivative is resistant to alpha-glucosidase and is suitable as a substrate for the alpha-amylase assay coupled with alpha-glucosidase in which the activity of alpha-amylase is determined by measuring the amount of p-nitrophenol liberated by alpha-glucosidase from p-nitrophenyl alpha-glucoside and p-nitrophenyl alpha-maltoside produced by the action of alpha-amylase. This alpha-amylase assay method was applied for determination of alpha-amylases in human serum.     

Studies on the substrate specificity of Taka-amylase A1. XIV. Preparation of 6-deoxy-6-halogenomaltotrioses and their hydrolysis by Taka-amylase A.

1. O-6-Deoxy-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose, O-6-chloro-6-deoxy-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose, O-6-bromo-6-deoxy-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose, and O-6-deoxy-6-iodo-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose were prepared, taking advantage of the substrate specificities of Taka-amylase A and glucoamylase, and the action of Taka-amylase A on these substrates was investigated. 2. The Michaelis constant Km and the molecular activity ko were determined at 37 degrees C and pH 5.2 using the modified maltotrioses. The values of Km and ko decreased upon modification of maltotriose and those of ko/Km were in agreement with the comparative initial rates for the corresponding derivatives of phenyl alpha-maltoside at low substrate concentrations. This result suggested that a subsite of the enzyme may have a specific interaction with halogen atoms in the substrate. 3. All halogenomaltotrioses examined showed substrate inhibition at high substrate concentrations.     

Separation of human salivary alpha-amylase isozymes by high-performance liquid chromatography with a continuous monitor system of the activity

Human salivary alpha-amylase isozymes were rapidly separated from each other by high-performance liquid chromatography with a postcolumn assay. The eluate from the HPLC column was mixed continuously with an intramolecularly quenched fluorescent substrate, p-nitro-phenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside delivered by a pump. The mixture was incubated in a reaction coil, and the fluorescence intensity was continuously measured by a fluorescence detector. The assay was based on the marked increase in fluorescence with the enzymatic cleavage of the glycosidic bond of the substrate that links the fluorogenic and quenching moieties.     

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

Aspergillus oryzae alpha-amylase [(1----4)-alpha-D-glucan glucanohydrolase, EC 3.2.1.1] produced O-(6-phosphoryl-alpha-D-glucopyranosyl)-(1----4)-O-alpha-D-glucopyran osy l-(1----4)-D-glucopyranose (6(3)-phosphorylmaltotriose) and O-alpha-D-glucopyranosyl-(1----4)-O-(3-phosphoryl-alpha-D-glucopyranosyl )- (1----4)-O-alpha-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)-alpha-D-glucan:orthophosphate alpha-D-glucosyltransferase, 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)-alpha-D-glucan, leaving three D-glucosyl residues attached to the 6-phosphorylated residue on the non-reducing side.     

Preparative use of the analytical column of a sugar autoanalyzer for resolution of gluco-oligosaccharides of the same molecular weight.

A sugar autoanalyzer was used on a preparative scale to resolve a gluco-oligosaccharide mixture. In this way the components of the following mixtures were resolved: O-alpha-D-glucopyranosyl-(1-3)-O-[alpha-D-glucopyranosyl-(1-6)]-D-glucose (1), O-alpha-D-glucopyranosyl-(1-6)-O-alpha-D-glucopyranosyl-(1-3)-D-glucose (2) and O-alpha-D-glucopyranosyl-(1-3)-O-alpha-D-glucopyranosyl-(1-6)-D-glucose (3), O-alpha-D-glucopyranosyl-(1-3)-O-alpha-D-glucopyranosyl-(1-4)-D-glucose (4) and O-alpha-D-glucopyranosyl-(1-4)-O-alpha-D-glucopyranosyl-(1-3)-D-glucose (5), and O-alpha-D-glucopyranosyl-(1-2)-O-alpha-D-glucopyranosyl-(1-6)-O-alpha-D-glucopranosyl-(1-6)-O-alpha-D-glucopyranosyl-(1-6)-D-glucose (6) and O-alpha-D-glucopyranosyl-(1-3)--O-alpha-D-glucopyranosyl-(1-6)-O-alpha-D-glucopyranosyl-(1-6)-O-alpha-D-glucopyranosyl-(1-6)-D-glucose (7).     

Examination of aglycone-binding site of human salivary alpha-amylase by means of transglycosylation reactions

The active site of human salivary alpha-amylase is composed of tandem subsites (S3, S2, S1, S1',S2', etc.) geometrically complementary to several glucose residues, and the glycosidic linkage of the substrate is split between S1 and S1'. As a matter of convenience, the subsites to which the non-reducing-end part (glycone) and the reducing-end part (aglycone) of the substrate being hydrolyzed are bound are named the glycone-binding site (S3, S2, S1) and the aglycone-binding site (S1', S2'), respectively. The features of the aglycone-binding site of human salivary alpha-amylase were examined by means of transglycosylation reaction using phenyl alpha-maltoside (GG phi: G-G-phi) and its derivatives (GAG phi: G-AG-phi, GCG phi: G-CG-phi, AGG phi: AG-G-phi, and CGG phi: CG-G-phi) in which one of the glucose residues (G) has been converted to 6-amino-6-deoxy-glucose (AG) or glucuronic acid (CG) residue as the acceptor. A fluorogenic derivative of maltotetraose, p-nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha-D -glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside (FG4P, FG-G-G-G-P), was used as the substrate. HSA catalyzed both hydrolysis of FG4P to FG3 (FG-G-G) and p-nitrophenyl alpha-glucoside (G-P) and transfer of the FG3 residue of FG4P to the acceptors. Transfer to GAG phi occurred more effectively than to GG phi. Transfers to GCG phi and CGG phi were less than to GG phi and very little transfer to AGG phi occurred.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the active site of Taka-amylase A: its action on phenyl maltooligosides with a charge at their non-reducing-ends

Five modified moltooligosaccharides, phenyl O-6-amino-6-deoxy-alpha-D- glucopyranosyl- (1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1-- --4)- alpha-D-glucopyransoide (AG4P), phenyl O-(alpha-D-glucopyranosyluronic acid)-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-d-glucopyran osy l- (1----4)-alpha-D-glucopyranoside (CG4P), phenyl O-6-amino-6-deoxy-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyra nos yl- (1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1-- --4)- alpha-D-glucopyranoside (AG5P), phenyl O-(alpha-D-glucopyranosyluronic acid)-(1----4)-O-alpha-D-glucopyranosyl- (1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1-- --4)- alpha-D-glucopyranoside (CG5P), and phenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)- O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-a lph a-D- glucopyranoside (FG4P), were prepared to examine the active site of Taka-amylase A (TAA) [EC 3.2.1.1, Aspergillus oryzae]. Phenyl alpha-maltotetraoside (G4P) was predominantly hydrolyzed by TAA to maltose and phenyl alpha-maltoside (G2P). While G2P, phenyl alpha-glucoside (GP), and phenol were liberated from AG4P in the ratio of 7:63:30. G4P, phenyl alpha-maltotrioside (G3P), G2P, and GP were liberated from G5P in the ratio of 1:20:73:6, but AG5P was almost completely hydrolyzed to modified maltotriose and G2P. On the hydrolysis of CG4P and CG5P, no remarkable change was observed except for a decrease in the relative reaction rates compared with G4P and G5P, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inspection of active sites of human salivary alpha-amylase isozymes by means of non-reducing-end substituted maltooligosaccharides with 2-pyridylamino residue

The modes of action of four alpha-amylase isozymes, which were purified from human saliva, on p-nitrophenyl alpha-maltopentaoside (G5P), maltohexaitol (G6R), and their 2-pyridylamino derivatives, p-nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha- D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside (FG5P) and O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)- O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O- alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-D- glucitol (FG6R) were examined at various pH values. No differences in their modes of action on the substrates was found. Irrespective of which enzyme was used, the molar ratio of the hydrolysis products of G5P or G6R was almost constant at any pH examined. On the other hand, those of FG5P and FG6R varied with pH such that predominantly O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl- (1----4)-O-alpha-D-glucopyranosyl-(1----4)-D-glucose (FG3) was formed at high pH ranges, while the formation of O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)- O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-D-gl ucose (FG4) increased at lower pH. The result indicates that the binding mode of FG5P or FG6R to the active sites of the enzymes changed with pH; namely, interactions between the 2-pyridylamino residue of the substrates and some amino acid residue(s) located in the active sites were influenced by pH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurement of cyclomaltodextrin glucanotransferase activity by high-performance liquid chromatography using a fluorogenic substrate

A mixture of p-nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranoside (FG5P) and p-nitrophenyl alpha-D-glucoside (GP) was incubated with cyclomaltodextrin glucanotransferase (CGTase) [EC 2.4.1.19]. Analysis of the digest by HPLC showed that the products were p-nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside (FG4P) and p-nitrophenyl alpha-D-maltoside (G2P), and no other product could be detected. Based on the reaction, a sensitive method to assay for CGTase was developed.     

Detection of human urinary alpha-amylase encoded by the AMY2B gene using a fluorogenic substrate, FG5P.

The existence of alpha-amylase (HXA) encoded by alpha-amylase gene AMY2B in healthy humans was examined using a fluorogenic substrate, FG5P (FG-G-G-G-G-P: FG, 6-deoxy-6-[(2-pyridyl)amino]-D-glucose residue; G, glucose residue; P, p-nitrophenyl residue; -, alpha-1,4-glycosidic bond). Chromatofocusing of urine from a healthy human was carried out. FG5P was digested with the fractions exhibiting alpha-amylase activity and each digest at an early stage was analyzed by HPLC. FG5P was hydrolyzed to FG3 (FG-G-G) and p-nitrophenyl alpha-maltoside (G-G-P), and to FG4 (FG-G-G-G) and p-nitrophenyl alpha-glucoside (G-P). The molar ratios of FG4 to FG3 (FG4/FG3) in the digests with basic fractions were larger than those in the digests of human pancreatic alpha-amylase (HPA, 1.11) and human salivary alpha-amylase (HSA, 0.51). Considering that the value for the AMY2B gene product with yeast (yHXA) is 1.88, a value of more than 1.11 implies that HXA exists. The amount of HXA was determined after removal of HSA on an anti-human salivary alpha-amylase antibody bound column. The FG4/FG3 values for six urine samples free from HSA were 1.23-1.26. Assuming that the FG4/FG3 value for HXA is the same as that for yHXA, the ratios of HXA and HPA were estimated to be 1:5.4-4.1. The results obtained showed that the AMY2B gene is usually expressed as HXA in healthy humans.     

Comparison of the action of glucoamylase and glucosyltransferase on D-glucose, maltose, and malto-oligosaccharides.

The action patterns of glucoamylase (amyloglucosidase) and glucosyltransferase (transglucosylase) on D-[1-14C]glucose, [1-14C]maltose, and [1-14C]malto-oligosaccharides (labeled at position 1 of the D-glucose group at the reducing end) have been investigated by paper-chromatographic and oligosaccharide-mapping techniques. Under the conditions of the experiments, the extent of conversion of D-glucose and of maltose into new oligosaccharides was 2.2 and 1.9% with glucoamylase, and 5.7 and 33% with glucosyltransferase. The major oligosaccharides produced by both enzymes were isomaltose (6-O-alpha-D-glucopyranosyl-alpha-D-glucose), panose (O-alpha-D-glucopyranosyl (1 leads to 6)-O-alpha-D-glucopyranosyl-(1 leads to 4)-alpha-D-glucose), and nigerose (3-O-alpha-D-glucopyranosyl-alpha-D-glucose). The glucosyltransferase also synthesized oligosaccharides from malto-oligosaccharides of higher molecular weight to yield compounds having alpha-(1 leads to 6)-linked D-glucosyl groups at the non-reducing ends. Glucoamylase exhibited little, if any, such activity on malto-oligosaccharides.     

Synthesis of structural elements of the capsular polysaccharides of Streptococcus pneumoniae types 6A and 6B

O-alpha-d-Glucopyranosyl-(1----3)-alpha, beta-L-rhamnopyranose (15), O-alpha-D-galactopyranosyl-(1----3)-O-alpha-D-glucopyranosyl-(1----3)-al pha, beta-L-rhamnopyranose (17), O-alpha-D-galactopyranosyl-(1----3)-O-alpha-D-glucopyranosyl-(1----3)- O-alpha-L-rhamnopyranosyl-(1----3)-D-ribitol (23), and O-alpha-D-galactopyranosyl-(1----3)-O-alpha-D-glucopyranosyl-(1----3)- O-alpha-L-rhamnopyranosyl-(1----4)-D-ribitol (27), which are structural elements of the capsular polysaccharides of Streptococcus pneumoniae types 6A and 6B ([----2)-alpha-D-Galp-(1----3)-alpha-D-Glcp-(1----3)-alpha-L-Rhap- (1----X)- D-Rib-ol-(5-P----]n; 6A X = 3, 6B X = 4), have been synthesised. Ethyl 3-O-allyl-2,4,6-tri-O-benzyl-1-thio-beta-D-glucopyranoside (3) was coupled with benzyl 2,4-di-O-benzyl-alpha-L-rhamnopyranoside (4), and subsequent deallylation (----14) and debenzylation gave 15. Condensation of 14 with ethyl 2,3,4,6-tetra-O-benzyl-1-thio-beta-D-galactopyranoside (2) followed by debenzylation gave 17. Acetylation of 17 followed by removal of AcO-1, conversion into the imidate, coupling with 1,2,4,5-tetra-O-benzyl-D-ribitol (11), deacetylation, and debenzylation gave 23. Coupling of the imidate with 1-O-allyloxycarbonyl-2,3,5-tri-O-benzyl-D-ribitol (12) followed by deallyloxycarbonylation, deacetylation, and debenzylation yielded 27.     

Hydrolysis of alpha-D-glucans and alpha-D-gluco-oligosaccharides by Cladosporium resinae glucoamylases

Culture filtrates of Cladosporium resinae ATCC 20495 contain a mixture of enzymes able to convert starch and pullulan efficiently into D-glucose. Culture conditions for optimal production of the pullulan-degrading activity have been established. The amylolytic enzyme preparation was fractionated by ion-exchange and molecular-sieve chromatography, and shown to contain alpha-D-glucosidase, alpha-amylase, and two glucoamylases. The glucoamylases have been purified to homogeneity and their substrate specificities investigated. One of the glucoamylases (termed P) readily hydrolyses the (1 leads to 6)-alpha-D linkages in pullulan, amylopectin, isomaltose, panose, and 6(3)-alpha-D-glucosylmaltotriose. Each of the glucoamylases cleaves the (1 leads to 6)-alpha-D linkage in panose much more readily than that in isomaltose.     

Structural examination of antitumour, water-soluble glucans from Grifora umbellata by use of four types of glucanase.

Antitumour glucans (GU) from the fungus Grifora umbellata have been subjected to periodate oxidation, Smith degradation, methylation analysis, and treatment with endo-(1 leads to 6)-beta-D-, endo-(1 leads to 3)-beta-D-, and exo-(1 leads to 3)-beta-D-glucanases, and alpha-amylase; the following structural features were revealed. GU-2 contains a backbone involving (1 leads to 6)-beta- and () leads to 3)-beta linkages, and two kinds of branches involving (1 leads to 6)-beta and (1 leads to 4)-alpha linkages. GU-3 has a (1 leads to 3)-beta-linked backbone and branches involving (1 leads to 6)-beta linkages or (1 leads to 4)-alpha and (1 leads to 6)-beta linkages. GU-4 also contains a (1 leads to 3) beta-D-glucan backbone and a small number of (1 leads to 6)-beta-linked branches. Probable structural units of these glucans are proposed.     

Synthesis of two phosphate-containing "heptasaccharide" fragments of the capsular polysaccharides of Streptococcus pneumoniae types 6A and 6B.

The "heptasaccharides" O-alpha-D-galactopyranosyl-(1----3)- O-alpha-D-glucopyranosyl-(1----3)-alpha, beta-L-rhamnopyranose 2'-[O-alpha-D-galactopyranosyl-(1----3)-O-alpha-D-glucopyranosyl- (1----3)-O-alpha-L-rhamnopyranosyl-(1----3)-D-ribit-5-yl sodium phosphate] (25) and O-alpha-D-galactopyranosyl- (1----3)-O-alpha-D-glucopyranosyl-(1----3)-alpha, beta-L-rhamnopyranose 2'-[O-alpha-D-galactopyranosyl-(1----3)-O-alpha-D-glucopyranosyl- (1----3)-O-alpha-L-rhamnopyranosyl-(1----4)-D-ribit-5-yl sodium phosphate] (27), which are structural elements of the capsular polysaccharides of Streptococcus pneumoniae types 6A and 6B ([----2)- -alpha-D-Galp-(1----3)-alpha-D-Glcp-(1----3)-alpha-L-Rhap- (1----X)-D-RibOH-(5-P----]n; 6A X = 3, 6B X = 4), respectively, have been synthesized. 2,4-Di-O-acetyl- 3-O-[2,4,6-tri-O-acetyl-3-O-(2,3,4,6-tetra-O-acetyl-alpha-D- galactopyranosyl)-alpha-D-glucopyranosyl]-alpha-L-rhamnopyranosyl trichloroacetimidate (13) was coupled with 5-O-allyloxycarbonyl-1,2,4-tri-O- benzyl-D-ribitol (10), using trimethylsilyl triflate as a promotor (----14), and deallyloxycarbonylation (----15) and conversion into the corresponding triethylammonium phosphonate then gave 16. Condensation of 16 with 4-methoxybenzyl 2,4-di-O-benzyl-3-O-[2,4,6-tri-O-benzyl-3-O-(3,4,6-tri-O-benzyl-alpha-D- galactopyranosyl)-alpha-D-glucopyranosyl]- alpha-L-rhamnopyranoside (22) followed by oxidation and deprotection afforded 25. 5-O-Allyl-1-O-allyloxycarbonyl-2,3-di-O-benzyl-D-ribitol (12) was coupled with 13, using trimethylsilyl triflate as a promoter, the resulting tetrasaccharide-alditol derivative 17 was deallyloxycarbonylated (----18), acetylated (----19), and deallylated (----20), and the product was converted into the triethylammonium phosphonate derivative 21. Condensation of 21 with 22 followed by oxidation and deprotection afforded 27.     

Kinetic investigation of a new inhibitor for human salivary alpha-amylase

This study is the first report on the effectiveness and specificity of alpha-acarviosinyl-(1-->4)-alpha-D-glucopyranosyl-(1-->6)-D-glucopyranosylidene-spiro-thiohydantoin (PTS-G-TH) inhibitor on the 2-chloro-4-nitrophenyl-4-O-beta-D-galactopyranosyl-maltoside (GalG2CNP) and amylose hydrolysis catalysed by human salivary alpha-amylase (HSA). Synthesis of PTS-G-TH was carried out by transglycosylation using acarbose as donor and glucopyranosylidene-spiro-thiohydantoin (G-TH) as acceptor. This new compound was found to be a much more efficient HSA inhibitor than G-TH. The inhibition is a mixed-noncompetitive type on both substrates and only one molecule of inhibitor binds to the enzyme. Kinetic constants calculated from secondary plots are in micromolar range. Values of K(EI) and K(ESI) are very similar in the presence of GalG2CNP substrate; 0.19 and 0.24 microM, respectively. Significant difference can be found for K(EI) and K(ESI) using amylose as substrate; 8.45 and 0.5 microM, respectively. These values indicate that inhibition is rather uncompetitive than competitive related to amylose hydrolysis.     

Asparaginyl glycopeptides with a low mannose content are hydrolyzed by endo-beta-N-acetylglucosaminidase H

Substrates susceptible to endo-beta-N-acetylglucosaminidase H were reduced in size through alpha-mannosidase treatment and periodate oxidation to yield the following compounds: (Man)4(GlcNAc)2Asn, [Manalpha 1 leads to 6Manalpha 1 leads to 6(Manalpha 1 leads to 3)Manbeta 1 leads to 4GlcNAcbeta 1 leads to 4GlcNACAsn]; (Man)3(GlcNAc)2Asn, [Manalpha 1 leads to 3Man-alpha 1 leads to 6Manbeta 1 leads to 4GlcNAcbeta 1 leads to 4GlcNAcAsn]; (Man)2(GlcNAc)2Asn, [Manalpha 1 leads to 6Manbeta1 leads to 4GlcNAcbeta 1 leads to 4BlcNAcAsm]. Comparison of the relative rates of hydrolysis of these compounds with (Man)5(GlcNAc)2-Asn, the most active substrate to date for the endoglycosidase, revealed (Man)4(GlcNAc)2Asn to be hydrolyzed faster than (Man)5(GlcNAc)2Asn and (Man)3-(GlcNAc)2Asn to be equal to or slightly better than (Man)5(GlcNAc)2Asn as a substrate. (Man)2(GlcNAc)2-Asn was completely hydrolyzed but at a rate that was about 10(4) slower than (Man)5(GlcNAc)2Asn, which is comparable to that for (Man)3(GlcNAc)2Asn(aa)x [Manalpha 1 leads to 6(Manalpha 1 leads to 3)Manbeta 1 leads to 4GlcNAcbeta 1 leads to 4GlcNAcAsn(aa)x], obtained from immunoglobulin M. (Man)1(GlcNAc)2Asn, [Manbeta 1 leads to 4GlcNAcbeta 1 leads to 4GlcNAcAsn] was hydrolyzed at a 100-fold slower rate than the latter glycopeptide. The effective range of endo-beta-N-acetylglucosaminidase H has thus been extended to compounds containing as few as 2 mannosyl residues.     

Studies on a thermostable alpha-amylase from the thermophilic fungus Scytalidium thermophilum

An alpha-amylase produced by Scytalidium thermophilum was purified using DEAE-cellulose and CM-cellulose ion exchange chromatography and Sepharose 6B gel filtration. The purified protein migrated as a single band in 6% PAGE and 7% SDS-PAGE. The estimated molecular mass was 36 kDa (SDS-PAGE) and 49 kDa (Sepharose 6B). Optima of pH and temperature were 6.0 and 60 degrees C, respectively. In the absence of substrate the purified alpha-amylase was stable for 1 h at 50 degrees C and had a half-life of 12 min at 60 degrees C, but was fully stable in the presence of starch. The enzyme was not activated by several metal ions tested, including Ca(2+) (up to 10 mM), but HgCl(2 )and CuCl(2) inhibited its activity. The alpha-amylase produced by S. thermophilum preferentially hydrolyzed starch, and to a lesser extent amylopectin, maltose, amylose and glycogen in that order. The products of starch hydrolysis (up to 6 h of reaction) analyzed by thin layer chromatography, showed oligosaccharides such as maltotrioses, maltotetraoses and maltopentaoses. Maltose and traces of glucose were formed only after 3 h of reaction. These results confirm the character of the enzyme studied to be an alpha-amylase (1,4-alpha-glucan glucanohydrolase).     

Some aspects of the mechanism of complexation of red kidney bean alpha-amylase inhibitor and alpha-amylase

Bovine pancreatic alpha-amylase binds 1 mol of acarbose (a carbohydrate alpha-amylase inhibitor) per mol at the active site and also binds acarbose nonspecifically. The red kidney bean alpha-amylase inhibitor-bovine pancreatic alpha-amylase complex retained nonspecific binding for acarbose only. Binding of p-nitrophenyl alpha-D-maltoside to the final complex of red kidney bean alpha-amylase inhibitor and bovine pancreatic alpha-amylase has a beta Ks (Ks') value that is 3.4-fold greater than the Ks (16 mM) of alpha-amylase for p-nitrophenyl alpha-D-maltoside alone. The initial complex of alpha-amylase and inhibitor apparently hydrolyzes this substrate as rapidly as alpha-amylase alone. The complex retains affinity for substrates and competitive inhibitors, which, when present in high concentrations, cause dissociation of the complex. Maltose (0.5 M), a competitive inhibitor of alpha-amylase, caused dissociation of the red kidney bean alpha-amylase inhibitor--alpha-amylase complex. Interaction between red kidney bean (Phaseolus vulgaris) alpha-amylase inhibitor and porcine pancreatic alpha-amylase proceeds through two steps. The first step has a Keq of 3.1 X 10(-5) M. The second step (unimolecular; first order) has a forward rate constant of 3.05 min-1 at pH 6.9 and 30 degrees C. alpha-Amylase inhibitor combines with alpha-amylase, in the presence of p-nitrophenyl alpha-D-maltoside, noncompetitively. On the basis of the data presented, it is likely that alpha-amylase is inactivated by the alpha-amylase inhibitor through a conformational change. A kinetic model, in the presence and absence of substrate, is described for noncompetitive, slow, tight-binding inhibitors that proceed through two steps.