Mechanism of maltal hydration catalyzed by beta-amylase: role of protein structure in controlling the steric outcome of reactions catalyzed by a glycosylase.

Crystalline (monomeric) soybean and (tetrameric) sweet potato beta-amylase were shown to catalyze the cis hydration of maltal (alpha-D-glucopyranosyl-2-deoxy-D-arabino-hex-1-enitol) to form beta-2-deoxymaltose. As reported earlier with the sweet potato enzyme, maltal hydration in D2O by soybean beta-amylase was found to exhibit an unusually large solvent deuterium kinetic isotope effect (VH/VD = 6.5), a reaction rate linearly dependent on the mole fraction of deuterium, and 2-deoxy-[2(a)-2H]maltose as product. These results indicate (for each beta-amylase) that protonation is the rate-limiting step in a reaction involving a nearly symmetric one-proton transition state and that maltal is specifically protonated from above the double bond. This is a different stereochemistry than reported for starch hydrolysis. With the hydration catalyzed in H2O and analyzed by gas-liquid chromatography, both sweet potato and soybean beta-amylase were found to convert maltal to the beta-anomer of 2-deoxymaltose. That maltal undergoes cis hydration provides evidence in support of a general-acid-catalyzed, carbonium ion mediated reaction. Of fundamental significance is that beta-amylase protonates maltal from a direction opposite that assumed for protonating starch, yet creates products of the same anomeric configuration from both. Such stereochemical dichotomy argues for the overriding role of protein structures in dictating the steric outcome of reactions catalyzed by a glycosylase, by limiting the approach and orientation of water or other acceptors to the reaction center.     

Scope and mechanism of carbohydrase action. Stereocomplementary hydrolytic and glucosyl-transferring actions of glucoamylase and glucodextranase with alpha- and beta-D-glucosyl fluoride

Rhizopus niveus glucoamylase and Arthrobacter globiformis glucodextranase, which catalyze the hydrolysis of starch and dextrans, respectively, to form D-glucose of inverted (beta) configuration, were found to convert both alpha- and beta-D-glucosyl fluoride to beta-D-glucose and hydrogen fluoride. Each enzyme directly hydrolyzes alpha-D-glucosyl fluoride but utilizes th beta-anomer in reactions that require 2 molecules of substrate and yield glucosyl transfer products which are then rapidly hydrolyzed to form beta-D-glucose. Various D-glucopyranosyl compounds serve as acceptors for such reactions. Mixtures of beta-D-glucosyl fluoride and methyl-alpha-D-glucopyranoside[14C], incubated with either enzyme, yielded both methyl-alpha-D-glucopyranosyl-(1 leads to 4)-alpha-D-[14C]glucopyranoside and methyl-alpha-D-glucopyranosyl-(1 leads to 6)-alpha-D-[14C]glucopyranoside. Glucoamylase produced more of the alpha-maltoside; glucodextranase produced more of the alpha-isomaltoside. Thus, both "exo-alpha-glucan hydrolases" emerge as glucosylases that catalyze stereospecifically complementary hydrolytic and transglucosylative reactions with glucosyl donors of opposite configuration. These reactions not only provide a new view of the catalytic capabilities of these supposedly strict hydrolases; they also furnish a basis for defining a detailed mechanism for catalysis. Present results, together with those of several recent studies from this laboratory (especially similar findings obtained with beta-amylase acting on alpha- and beta-maltosyl fluoride (Hehre, E. J., Brewer, C. F., and Genghof, D. S. (1979) J. Biol. Chem. 254, 5942-5950), provide strong new evidence for the functional flexibility of the catalytic groups of carbohydrases.     

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)     

Action of neopullulanase. Neopullulanase catalyzes both hydrolysis and transglycosylation at alpha-(1----4)- and alpha-(1----6)-glucosidic linkages.

The transglycosylation reaction catalyzed by neopullulanase was analyzed. Radioactive oligosaccharides were produced when the enzyme acted on maltotriose in the presence of [U-14C]glucose. Some of the radioactive oligosaccharides had only alpha-(1----4)-glucosidic linkages, but others were suggested to have alpha-(1----6)-glucosidic linkages. The existence of alpha-(1----6)-glucosidic linkages in the products from maltotriose with neopullulanase was proven by proton NMR spectroscopy and methylation analysis. We previously reported that the one active center of neopullulanase catalyzes the hydrolysis of alpha-(1----4)- and alpha-(1----6)-glucosidic linkages (Kuriki, T., Takata, H., Okada, S., and Imanaka, T. (1991) J. Bacteriol. 173,6147-6152). These facts proved that neopullulanase catalyzed all four types of reactions: hydrolysis of alpha-(1----4)-glucosidic linkage, hydrolysis of alpha-(1----6)-glucosidic linkage, transglycosylation to form alpha-(1----4)-glucosidic linkage, and transglycosylation to form alpha-(1----6)-glucosidic linkage. The four reactions are typically catalyzed by alpha-amylase, pullulanase, cyclomaltodextrin glucanotransferase, and 1,4-alpha-D-glucan branching enzyme, respectively. These four enzymes have some structural similarities to one other, but reactions catalyzed by the enzymes are considered to be distinctive: the four reactions are individually catalyzed by each of the enzymes. The experimental results obtained from the analysis of the reaction of the neopullulanase exhibited that the four reactions can be catalyzed in the same mechanism.     

Factors determining steric course of enzymic glycosylation reactions: glycosyl transfer products formed from 2,6-anhydro-1-deoxy-D-gluco-hept-1-enitol by alpha-glucosidases and an inverting exo-alpha-glucanase

Glycosyl transfer products were formed from 2,6-anhydro-1-deoxy-D-gluco-hept-1-enitol (heptenitol) by purified alpha-glucosidases from Candida tropicalis and rice and by an inverting exo-alpha-glucanase (glucodextranase) from Arthrobacter globiformis. The products were structurally defined through 1H and 13C NMR (nuclear magnetic resonance) spectra of their crystalline per-O-acetates in comparison with those of authentic methyl 1-deoxy-alpha- and methyl 1-deoxy-beta-D-gluco-heptuloside. 1-Deoxy-alpha-D-gluco-heptulosyl-(2 leads to 7)-heptenitol and 1-deoxy-alpha-D-gluco-heptulosyl-(2 leads to 7)-D-gluco-heptulose were produced by both the Candida alpha-glucosidase and the glucodextranase; 1-deoxy-alpha-D-gluco-heptulosyl-(2 leads to 5)- and 1-deoxy-alpha-D-gluco-heptulosyl-(2 leads to 7)-D-gluco-heptuloses by the rice alpha-glucosidase. These results, together with our earlier findings of sterospecific hydration of heptenitol catalyzed by the same enzymes [Hehre, E. J., Brewer, C. F., Uchiyama, T., Schlesselmann, P., & Lehmann, J. (1980) Biochemistry 19, 3557-3564], show the inadequacy of the long-accepted notion that carbohydrase-catalyzed reactions always lead to retention (or always lead to inversion) of substrate configuration. In particular, the finding that glucodextranase forms transfer products of alpha configuration and a hydration product of beta configuration from the same substrate provides a clear example of the functioning of acceptors rather than donor substrates in selecting the steric course of reactions catalyzed by a glycosylase. The circumstances under which acceptor cosubstrates might be expected to show this significant effect are discussed. The opportunity presumably would exist whenever carbonium ion mediated reactions are catalyzed by glycosylases that provide oppositely oriented approaches of different acceptors to the catalytic center.     

Stereochemistry of D-galactal and D-galacto-octenitol hydration by coffee bean alpha-galactosidase: insight into catalytic functioning of the enzyme.

Green coffee bean alpha-galactosidase was found to catalyze the hydration of D-galactal and (Z)-3,7-anhydro-1,2-dideoxy-D-galacto-oct-2-enitol (D-galacto-octenitol), each a known substrate for beta-galactosidase. The hydration of D-galactal by the alpha-galactosidase in D2O yielded 2-deoxy-2(S)-D-[2-2H]galactose; the hydration of D-[2-2H]galacto-octenitol in H2O yielded 1,2-dideoxy-2(R)-D-[2-2H]galactooct-3-ulose. Thus, the enzyme protonated each substrate from beneath the plane of the ring, as assumed for alpha-D-galactosides. These results provide an unequivocal assignment of the orientation of an acidic catalytic group to the alpha-galactosidase reaction center. In addition, they reveal a pattern of glycal/exocyclic enitol/glycoside protonation by the enzyme that differs from the pattern reported for beta-galactosidase and from that reported for alpha-glucosidases. Further findings show that D-galacto-octenitol is hydrated by the coffee bean alpha-galactosidase to form the alpha-anomer of 1,2-dideoxy-D-galactooctulose and by Escherichia coli beta-galactosidase to form the beta-anomer. That each enzyme converts this enolic substrate to a product whose de novo anomeric configuration matches that formed from its D-galactosidic substrates provides new evidence for the role of protein structure in controlling the steric outcome of reactions catalyzed by these and other glycosylases. The findings are discussed in light of the concept that catalysis by glycosidases involves a "plastic" protonation phase and a "conserved" product configuration phase.     

Hydration of cellobial by exo- and endo-type cellulases: evidence for catalytic flexibility of glycosylases

New insight has been obtained into the catalytic capabilities of cellulase. Essentially homogeneous preparations of exo- (or Avicelase-) type and endo- (or CMCase-) type cellulases from Irpex lacteus and Aspergillus niger, respectively, were shown to hydrate the enolic bond of cellobial to form 2-deoxycellobiose. The A. niger enzyme also synthesized a small amount of a 2-deoxycellobiosyl-transfer product from cellobial. By use of digests conducted in deuterated buffer and 1H NMR spectra for product analysis, both cellulases were found to protonate (deuterate) the double bond of cellobial from below the si face of the D-glucal moiety, i.e., from a direction opposite that assumed for protonation of the beta-D-glycosidic linkages of cellulose and cellodextrins. The exo enzyme, which hydrolyzes the latter substrates primarily to cellobiose, rapidly catalyzed cellobial hydration to produce the beta-anomer of beta-D-glucopyranosyl(1----4)-2-deoxy-D-glucose-2(e)-d. The A. niger cellulase produced the same 2-deoxycellobiose-d from cellobial, though too slowly for its configuration to be determined. However, evidence was obtained for the formation of a beta-2-deoxycellobiosyl-d-D-glucose-transfer product by the enzyme. Thus, it is likely that all of the observed reactions with cellobial represent trans additions at the double bond. In any case, the anomeric configuration of products is created de novo. Separate mechanisms are described for the reaction of cellobial hydration and for the stereochemically different reaction of cellulose hydrolysis catalyzed by the present enzymes, assuming an arrangement of their catalytic groups analogous to that found in lysozyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of beta(1----3)glucan synthetase of various fungi by nucleoside triphosphates: generalized regulatory mechanism for cell wall biosynthesis.

Particulate fractions from the taxonomically diverse fungi Achlya ambisexualis, Hansenula anomala, Neurospora crassa, Cryptococcus laurentii, Schizophyllum commune, and Wangiella dermatitidis were found to catalyze the time-dependent incorporation of glucose from UDP-[14C]glucose into a water-insoluble material. The reaction was stimulated by bovine serum albumin. The product was characterized as beta(1----3)glucan on the basis of its resistance to alpha- and beta-amylase and susceptibility to beta(1----3)glucanase. With the exception of the preparation from A. ambisexualis, all others were stimulated by nucleoside triphosphates and their analogs. The best activators were GTP and guanosine 5'-(gamma-thio)triphosphate. It is concluded that the stimulation by nucleotides, previously found with the glucan synthetase of Saccharomyces cerevisiae, is a regulatory mechanism that was well conserved during fungal evolution, presumably because of its importance in controlling cell wall biosynthesis and cell growth.     

Hydrolysis of aryl beta-maltotriosides by sweet potato beta-amylase and soybean beta-amylase.

Sweet potato beta-amylase [EC 3.2.1.2, alpha 1,4-D-glucan maltohydrolase]-catalyzed hydrolyses of aryl beta-maltotriosides with substituents, NO2-, Cl-, and Br- at the o-, m-, and p-positions in the phenyl ring were studied at pH 4.8 and 25 degrees C. The hydrolyses of a few of the maltotriosides by soybean beta-amylase [EC 3.2.1.2, alpha-1,4-D-glucan maltohydrolase] were also studied at pH 5.4 and 25 degrees C. It was found that the aryl beta-maltotriosides were preferentially hydrolyzed into maltose and aryl beta-D-glucosides by both beta-amylases. The Michaelis constant Km and the molecular activity ko were determined for the hydrolyses of these maltotriosides and compared with those of maltotriose and maltotetraose. Aryl beta-maltotriosides were more rapidly hydrolyzed than maltotriose by a factor of 30--80, and more slowly hydrolyzed than maltotetraose by a factor of 10--30, depending on the kinds of substituents. The rapid hydrolysis of aryl beta-maltotrioside as compared with maltotriose may be due to the interaction of an aryl group with the subsite of beta-amylase. This is in contrast with glucoamylase [EC 3.2.1.3, alpha-1,4-D-glucan glucohydrolase] of Rhizopus niveus-catalyzed hydrolysis of phenyl beta-maltoside, whose phenyl group does not interact so much with the subsite of the enzyme.     

Structural and enzymatic analysis of soybean beta-amylase mutants with increased pH optimum

Comparison of the architecture around the active site of soybean beta-amylase and Bacillus cereus beta-amylase showed that the hydrogen bond networks (Glu380-(Lys295-Met51) and Glu380-Asn340-Glu178) in soybean beta-amylase around the base catalytic residue, Glu380, seem to contribute to the lower pH optimum of soybean beta-amylase. To convert the pH optimum of soybean beta-amylase (pH 5.4) to that of the bacterial type enzyme (pH 6.7), three mutants of soybean beta-amylase, M51T, E178Y, and N340T, were constructed such that the hydrogen bond networks were removed by site-directed mutagenesis. The kinetic analysis showed that the pH optimum of all mutants shifted dramatically to a neutral pH (range, from 5.4 to 6.0-6.6). The Km values of the mutants were almost the same as that of soybean beta-amylase except in the case of M51T, while the Vmax values of all mutants were low compared with that of soybean beta-amylase. The crystal structure analysis of the wild type-maltose and mutant-maltose complexes showed that the direct hydrogen bond between Glu380 and Asn340 was completely disrupted in the mutants M51T, E178Y, and N340T. In the case of M51T, the hydrogen bond between Glu380 and Lys295 was also disrupted. These results indicated that the reduced pKa value of Glu380 is stabilized by the hydrogen bond network and is responsible for the lower pH optimum of soybean beta-amylase compared with that of the bacterial beta-amylase.     

17O-NMR studies of the conformational and dynamic properties of enkephalins in aqueous and organic solutions using selectively labeled analogues

The synthesis of Leu-enkephalin selectively 17O-enriched in Gly2 and Gly3 is reported. The 17O-nmr chemical shifts of [17O-Gly2, Leu5]- and [17O-Gly3, Leu5]-enkephalins in H2O are almost identical and independent of the pH. Since hydrogen bonding is the dominant factor governing the chemical shifts of the peptide oxygen, it can be concluded that the hydration state of both oxygens is identical and independent of the pH. The 17O chemical shifts of the [17O-Leu5]-enkephalin terminal carboxyl group at pH approximately 1.9 and 5.6 are very different in H2O but very similar in CH3CN/DMSO (4:1) solution. This suggests that the protonation state of the carboxyl group at both pH values in CH3CN/DMSO solution is the same and consequently that Leu-enkephalin exists in the neutral form at pH approximately 5.6. In this organic mixed solvent system both Gly2 and Gly3 oxygen resonances exhibit a significant shift to high frequency by the same extent (delta delta approximately 30 ppm). It is concluded that both peptide oxygens are not hydrogen bonded to an appreciable extent and that no specific 2----5 hydrogen bonding exists to an appreciable extent. This conclusion is in agreement with the energy of activation for molecular rotation, as determined from T1 measurements, which was found to be almost identical for both [17O-Gly2, Leu5]- and [17O-Gly3, Leu5]-enkephalins in CH3CN/DMSO (4:1) mixed solvent.     

Purification and determination of the structure of capsular polysaccharide of Vibrio vulnificus M06-24.

Virulence of Vibrio vulnificus has been strongly associated with encapsulation and an opaque colony morphology. Capsular polysaccharide was purified from a whole-cell, phosphate-buffered saline-extracted preparation of the opaque, virulent phase of V. vulnificus M06-24 (M06-24/O) by dialysis, centrifugation, enzymatic digestion, and phenol-chloroform extraction. Nuclear magnetic resonance spectroscopic analysis of the purified polysaccharide showed that the polymer was composed of a repeating structure with four sugar residues per repeating subunit: three residues of 2-acetamido-2,6-dideoxyhexopyranose in the alpha-gluco configuration (QuiNAc) and an additional residue of 2-acetamido hexouronate in the alpha-galactopyranose configuration (GalNAcA). The complete carbohydrate structure of the polysaccharide was determined by heteronuclear nuclear magnetic resonance spectroscopy and by high-performance anion-exchange chromatography. The 1H and 13C nuclear magnetic resonance spectra were completely assigned, and vicinal coupling relationships were used to establish the stereochemistry of each sugar residue, its anomeric configuration, and the positions of the glycosidic linkages. The complete structure is: [----3) QuipNAc alpha-(1----3)-GalpNAcA alpha-(1----3)-QuipNAc alpha-(1----]n QuipNAc alpha-(1----4)-increases The polysaccharide was produced by a translucent phase variant of M06-24 (M06-24/T) but not by a translucent, acapsular transposon mutant (CVD752). Antibodies to the polysaccharide were demonstrable in serum from rabbits inoculated with M06-24/O.     

Steric course of the hydration of D-gluco-octenitol catalyzed by alpha-glucosidases and by trehalase

Crystalline Aspergillus niger alpha-glucosidase and highly purified preparations of rice alpha-glucosidase II and Trichoderma reesei trehalase were found to catalyze the hydration of [2-(2)H]-D-gluco-octenitol, i.e., (Z)-3,7-anhydro-1,2-dideoxy-[2-2H]-D-gluco-oct-2-enitol, to yield 1,2-dideoxy-[2-2H]-D-gluco-octulose. In each case, the stereochemistry of the reaction was elucidated by examining the newly formed centers of asymmetry at C-2 and C-3 of the hydration product. The C-1 to C-3 fragment of each isolated [2-2H]-D-gluco-octulose product was recovered as [2-2H]propionic acid and identified by its positive optical rotatory dispersion as the S isomer, showing that each enzyme had protonated the octenitol (at C-2) from above its re face. 1H NMR spectra of enzyme/D-gluco-octenitol digests in D2O showed that the alpha-anomer of [2-2H]-D-gluco-octulose was exclusively produced by each alpha-glucosidase, whereas the beta-anomer was formed by action of the trehalase. The trans hydration catalyzed by the alpha-glucosidases was found to be very strongly inhibited by the substrate; the cis hydration reaction catalyzed by the trehalase showed no such inhibition. Special importance is attached to the finding that in hydrating octenitol each enzyme creates a product of the same anomeric form as in hydrolyzing an alpha-D-glucosidic substrate. This result adds substantially to the growing evidence that individual glycosylases create the configuration of their reaction products by a means that is independent of donor substrate configuration, that is, by a means other than "retaining" or "inverting" substrate configuration.     

Purification and characterization of alpha-L-fucosidase from Bacillus circulans grown on porcine gastric mucin.

Bacillus circulans isolated from soil was found to produce two types of alpha-L-fucosidase differing in substrate specificity. One was able to liberate L-fucose from porcine gastric mucin (PGM), but not from artificial substrates, including p-nitrophenyl and methyl alpha-L-fucosides, while the other acted not on PGM but on p-nitrophenyl alpha-L-fucoside. The production of the former enzyme was enhanced about 150 times as much by PGM added to the medium as by glucose. The alpha-L-fucosidase acting on PGM was purified from the culture fluid obtained with PGM medium by ammonium sulfate fractionation and subsequent column chromatography. The purified enzyme was found to be homogeneous by PAGE and its molecular weight was estimated to be approximately 285,000. The optimum pH was found to be 5.5 to 6.5 and the stable pH range was 4.5 to 9.0. The enzyme decomposed various blood group O(H) active substances such as PGM, human milk and human saliva, and moreover acted on A-, B-, and O-erythrocytes. The enzyme was shown to cleave alpha-(1----2)-, (1----3)-, and (1----4)-L-fucosidic linkages in various glycoproteins and oligosaccharides, but failed to hydrolyze alpha-(1----6)-L-fucosic linkages in 6-O-alpha-L-fucopyranosyl-N-acetylglucosamine and intact bovine thyroglobulin.     

Reverse protonation is the key to general acid-base catalysis in enolase

The pH dependence of enolase catalysis was studied to understand how enolase is able to utilize both general acid and general base catalysis in each direction of the reaction at near-neutral pHs. Wild-type enolase from yeast was assayed in the dehydration reaction (2-phospho-D-glycerate --> phosphoenolpyruvate + H(2)O) at different pHs. E211Q, a site-specific variant of enolase that catalyzes the exchange of the alpha-proton of 2-phospho-D-glycerate but not the complete dehydration, was assayed in a (1)H/(2)H exchange reaction at different pDs. Additionally, crystal structures of E211Q and E168Q were obtained at 2.0 and 1.8 A resolution, respectively. Analysis of the pH profile of k(cat)/K(Mg) for wild-type enolase yielded macroscopic pK(a) estimates of 7.4 +/- 0.3 and 9.0 +/- 0.3, while the results of the pD profile of the exchange reaction of E211Q led to a pK(a) estimate of 9.5 +/- 0.1. These values permit estimates of the four microscopic pK(a)s that describe the four relevant protonation states of the acid/base catalytic groups in the active site. The analysis indicates that the dehydration reaction is catalyzed by a small fraction of enzyme that is reverse-protonated (i.e., Lys345-NH(2), Glu211-COOH), whereas the hydration reaction is catalyzed by a larger fraction of the enzyme that is typically protonated (i.e., Lys345-NH(3)(+), Glu211-COO(-)). These two forms of the enzyme coexist in a constant, pH-independent ratio. The structures of E211Q and E168Q both show virtually identical folds and active-site architectures (as compared to wild-type enolase) and thus provide additional support to the conclusions reported herein. Other enzymes that require both general acid and general base catalysis likely require reverse protonation of catalytic groups in one direction of the reaction.     

Engineering of the pH optimum of Bacillus cereus beta-amylase: conversion of the pH optimum from a bacterial type to a higher-plant type

The optimum pH of Bacillus cereus beta-amylase (BCB, pH 6.7) differs from that of soybean beta-amylase (SBA, pH 5.4) due to the substitution of a few amino acid residues near the catalytic base residue (Glu 380 in SBA and Glu 367 in BCB). To explore the mechanism for controlling the optimum pH of beta-amylase, five mutants of BCB (Y164E, Y164F, Y164H, Y164Q, and Y164Q/T47M/Y164E/T328N) were constructed and characterized with respect to enzymatic properties and X-ray structural crystal analysis. The optimum pH of the four single mutants shifted to 4.2-4.8, approximately 2 pH units and approximately 1 pH unit lower than those of BCB and SBA, respectively, and their k(cat) values decreased to 41-3% of that of the wild-type enzyme. The X-ray crystal analysis of the enzyme-maltose complexes showed that Glu 367 of the wild type is surrounded by two water molecules (W1 and W2) that are not found in SBA. W1 is hydrogen-bonded to both side chains of Glu 367 and Tyr 164. The mutation of Tyr 164 to Glu and Phe resulted in the disruption of the hydrogen bond between Tyr 164 Oeta and W1 and the introduction of two additional water molecules near position 164. In contrast, the triple mutant of BCB with a slightly decreased pH optimum at pH 6.0 has no water molecules (W1 and W2) around Glu 367. These results suggested that a water-mediated hydrogen bond network (Glu 367...W1...Tyr 164...Thr 328) is the primary requisite for the increased pH optimum of wild-type BCB. This strategy is completely different from that of SBA, in which a hydrogen bond network (Glu 380...Thr 340...Glu 178) reduces the optimum pH in a hydrophobic environment.     

New mannose-specific lectins from garlic (Allium sativum) and ramsons (Allium ursinum) bulbs.

Two new mannose-binding lectins were isolated from garlic (Allium sativum, ASA) and ramsons (Allium ursinum, AUA) bulbs, of the family Alliaceae, by affinity chromatography on immobilized mannose. The carbohydrate-binding specificity of these two lectins was studied by quantitative precipitation and hapten-inhibition assay. ASA reacted strongly with a synthetic linear (1----3)-alpha-D-mannan and S. cerevisiae mannan, weakly with a synthetic (1----6)-alpha-D-mannan, and failed to precipitate with galactomannans from T. gropengiesseri and T. lactis-condensi, a linear mannopentaose, and murine IgM. On the other hand, AUA gave a strong reaction of precipitation with murine IgM, and good reactions with S. cerevisiae mannan and both synthetic linear mannans, suggesting that the two lectins have somewhat different binding specificities for alpha-D-mannosyl units. Of the saccharides tested as inhibitors of precipitation, those with alpha-(1----3)-linked mannosyl units were the best inhibitors of ASA, the alpha-(1----2)-, alpha-(1----4)-, and alpha-(1----6)-linked mannobioses and biosides having less than one eighth the affinity of the alpha-(1----3)-linked compounds. The N-terminal amino acid sequence of ASA exhibits 79% homology with that of AUA, and moderately high homology (53%) with that of snowdrop bulb lectin, also an alpha-D-mannosyl-binding lectin.     

Biosynthesis of disialylated beta-D-galactopyranosyl-(1----3)-2-acetamido-2-deoxy-beta-D-glucopy ran osyl oligosaccharide chains. Identification of a beta-D-galactoside alpha-(2----3)- and a 2-acetamido-2-deoxy-beta-D-glucoside alpha-(2----6)-sialyltransferase in regenerating rat liver and other tissues

Regenerating rat liver microsomes contain a beta-D-galactoside alpha-(2----3)- and a 2-acetamido-2-deoxy-beta-D-glucoside alpha-(2----6)-sialyltransferase that are involved in the synthesis of the terminal alpha-NeuAc-(2----3)-beta-D-Galp-(1----3)-alpha-[NeuAc-(2----6)]-beta- D-GlcpNAc-(1----R) group occurring in human milk oligosaccharides and the glycan chains of several N-glycoproteins. Analysis by liquid chromatography and methylation of the products of sialylation obtained when lacto-N-tetraose [beta-D-Galp-(1----3)-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4) -D-Glc] was used as a substrate in the incubations in vitro indicated that the disialylated sequence is formed for greater than 95% through the tetrasaccharide alpha-NeuAc-(2----3)-beta-D-Gal-(1----3)-beta-D-GlcNAc-(1----3)-beta-D-G al- (1----4)-D-Glc as one of two possible intermediates. This indicates that in the synthesis of the disialylated sequence the alpha-(2----3)- and the alpha-(2----6)-sialyltransferase act in a highly preferred order in which the alpha-(2----3) enzyme acts first. This order is imposed by the specificity of the alpha-(2----6)-sialyltransferase, which requires an alpha-NeuAc-(2----3)-beta-D-Gal-(1----3)-beta-D-GlcNAc-(1----R) sequence for optimal activity, and shows very low and no activity with beta-D-Gal-(1----3)-beta-D-GlcNAc-(1----R) and beta-D-GlcNAc-(1----R) acceptor structures, respectively. Results obtained with normal rat, fetal calf, rabbit and human liver, and human placenta indicated that very similar or identical sialyltransferases occur in these tissues. It is suggested that these enzymes differ from the sialyltransferases that previously had been identified in fetal calf liver and human placenta.     

Biosynthesis of the blood group Pk and P1 antigens by human kidney microsomes.

On human erythrocytes, the membrane components associated with Pk and P1 blood-group specificity are glycosphingolipids that carry a common terminal alpha-D-Galp-(1----4)-beta-D-Gal unit, the biosynthesis of which is poorly understood. Human kidneys typed for P1 and P2 (non-P1) blood-group specificity have been assayed for (1----4)-alpha-D-galactosyltransferase activity by use of lactosylceramide [beta-D-Galp-(1----4)-beta-D-Glcp-ceramide] and paragloboside [beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-beta-D-Galp- (1----4)-beta-D-Glcp-ceramide] as acceptor substrates. The linkage and anomeric configuration of the galactosyl group transferred into the reaction products were established by methylation analysis before and after alpha- and beta-D-galactosidase treatments, as well as by immunostaining using specific monoclonal antibodies directed against the Pk and P1 antigens. The results demonstrated that the microsomal proteins from P1 kidneys catalyze the synthesis of Pk [alpha-D-Galp-(1----4)-beta-D-Galp-(1----4)-beta-D-Glcp-ceramide] and P1 [alpha-D-Galp-(1----4)-beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-beta -D-Galp-(1----4)-beta-D-Glcp-ceramide] glycolipids, whereas microsomes from P2 kidney catalyze the synthesis of the Pk glycolipid, but not of the P1 glycolipid. Competition studies using a mixture of two oligosaccharides (methyl beta-lactoside and methyl beta-lacto-N- neotetraoside) or of two glycolipids (lactosylceramide and paragloboside) as acceptors indicated that these substrates do not compete for the same enzyme in the microsomal preparation from P1 kidneys. The results suggested that the Pk and P1 glycolipids are synthesized by two distinct enzymes.     

Purification and some properties of a novel alpha-L-fucosidase capable of acting on alpha-(1----6)-L-fucosidic linkages from Bacillus circulans M28

Two types of alpha-L-fucosidase (F-I and F-II), that differ in substrate specificity, were produced in the culture fluid by Bacillus circulans isolated from soil when the bacterium was cultivated on medium containing porcine gastric mucin. F-I was able to cleave the alpha-(1----2), alpha-(1----3), and alpha-(1----4)-L-fucosidic linkages in various oligosaccharides and glycoproteins, but not p-nitrophenyl alpha-L-fucoside, as previously reported [Y. Tsuji et al. (1990) J. Biochem. 107, 324-330]. F-II was purified from the culture fluid obtained with glucose medium by ammonium sulfate fractionation and various subsequent column chromatographies. The purified enzyme was found to be homogeneous on PAGE and its molecular weight was estimated to be approximately 250,000. The maximal activity was observed between pH 6.0 to 7.0, the stable pH range being 6.0 to 8.5. The enzyme specifically cleaved alpha-L-fucosidic bonds in low molecular weight substrates. The enzyme cleaved not only p-nitrophenyl alpha-L-fucoside, but also 2-fucosyllactose and 3-fucosyllactose. The enzyme was also able to act on the alpha-(1----6)-L-fucosidic linkages to N-acetylglucosamine in 6-O-alpha-L-fucopyranosyl-N-acetylglucosamine, and bi- and tetra-antennary oligosaccharides derived from porcine pancreatic lipase, which were not hydrolyzed by F-I.