Biomedical subjects

C F Brewer

Publications and source records attributed to C F Brewer.

At least 37 records · Page 2Linked to original sources

PubMed1994

Homogeneous aggregation of the 14-kDa beta-galactoside specific vertebrate lectin complex with asialofetuin in mixed systems.

The galactose-specific 14-kDa family of animals lectins are an evolutionary conserved group of proteins that have been implicated in a wide variety of biological processes including cell proliferation, adhesion, and transformation. The present study demonstrates that the dimeric 14-kDa calf spleen lectin forms homogeneous aggregated cross-linked complexes with asialofetuin, a glycoprotein with multiple carbohydrate chains possessing terminal galactose residues, in the presence of other lectins with similar specificities and cross-linking activities. Several galactose-specific plant lectins also form homogeneous aggregated cross-linked complexes with ASF. These results demonstrate a new source of specificity for the 14-kDa family of vertebrate lectins, namely, the ability to selectively cross-link and aggregate glycoproteins in mixed systems. The results have important biological implications for the interactions of multivalent lectins and glycoconjugates, as well as the thermodynamic interactions of multivalent molecules in general.

Animals↗

PubMed1994

Observation of unique cross-linked lattices between multiantennary carbohydrates and soybean lectin. Presence of pseudo-2-fold axes of symmetry in complex type carbohydrates.

The tetrameric lectin from Glycine max (soybean) (SBA) has been shown to cross-link and precipitate with N-linked multiantennary complex type oligosaccharides containing nonreducing terminal Gal residues (Bhattacharyya, L., Haraldsson, M., & Brewer, C. F. (1988) Biochemistry 27, 1034-1041). In the present study, negative stain electron micrographs of the precipitates of SBA with a series of naturally occurring and synthetic multiantennary carbohydrates with terminal Gal or GalNAc residues show the presence of highly ordered cross-linked lattices for many of the complexes. The precipitates of SBA with a "bisected" and "nonbisected" N-linked biantennary complex type oligosaccharide containing Gal residues at the nonreducing termini show similar two-dimensional patterns. However, the pattern observed for the precipitates of a tetraantennary complex type oligosaccharide with SBA is distinct from those of the two biantennary carbohydrates. Furthermore, the precipitates formed between the lectin and a synthetic O-linked biantennary ("cluster") glycoside with terminal GalNAc residues show a pattern that is different from those above. Four biantennary pentasaccharide analogs of the blood group I antigen containing beta-LacNAc moieties at the 2.3-, 2.4-, 2.6-, and 3.6-positions of the core Gal also showed ordered patterns in their precipitates with SBA. X-ray crystallographic data and mixed quantitative precipitation profiles of binary mixtures of the four analogs demonstrate that each analog possesses a unique cross-linked lattice with the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed1994

Purification and characterization of three isolectins of soybean agglutinin. Evidence for C-terminal truncation by electrospray ionization mass spectrometry.

Soybean agglutinin (SBA) is a tetrameric D-Gal/D-GalNAc-specific lectin possessing one Man9 oligomannose-type chain/monomer. SBA exists as multiple isolectins having similar binding and immunochemical properties. The present study shows that native SBA consists of at least five isolectins. Three of these isoforms have been purified by chromatofocusing and designated as SBA-I, SBA-II and SBA-III in order of their elution from a chromatofocusing column. The pI of the isolectins are 7.0, 6.85 and 6.7, respectively, as determined by isoelectric focusing. Each isolectin was denatured in 6 M guanidine hydrochloride into their individual subunits which were separated by reverse-phase high performance liquid chromatography (RP-HPLC). The HPLC profiles were similar for all three isoforms which showed two major peaks (peak 1 and peak 3) along with a minor peak (peak 2). The first peak of SBA-II existed as a double labeled as 1 a and 1 b. Each peak was analyzed by electrospray ionization mass spectrometry to characterize each isoform and determine their structural differences. The calculated mass of an intact lectin monomer from the amino acid sequence (253 residues) derived from cDNA of the lectin including a Man9 oligomannose chain is 29438 Da. The present results show that peak 3 of each isoform corresponds to an intact subunit (alpha) while peak 1 of each isoform shows lower masses which are assigned to C-terminal fragmentation of the protein. Peak 1 of SBA-I has a molecular mass of 28000Da corresponding to a fragmented subunit (beta) consisting of 240 residues (calculated molecular mass 28001Da). Peak 1a of SBA-II shows a molecular mass of 28000Da corresponding to a fragmented beta subunit, while peak 1b showed two major species: a 28000-Da (beta subunit) and a 28327-Da subunit which corresponds to 243 residues (calculated mass 28326Da) designated as a gamma subunit. In addition, peak 1b showed the presence of a molecular species of 28627Da corresponding to a 246-residue subunit (gamma'). Peak 1 of SBA-III showed a major molecular species corresponding to a fragmented gamma subunit. The minor peak in the HPLC profile (peak 2) represented a subunit of 252 residues for all three isoforms. The results suggest that the subunit compositions of SBA-I, SBA-II and SBA-III are approximately alpha 2 beta 2, alpha 2 beta gamma and alpha 2 gamma 2, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1994

Differences in the cross-linking activities of native and recombinant Erythrina corallodendron lectin with asialofetuin. Evidence for carbohydrate-carbohydrate interactions in lectin-glycoprotein complexes.

A previous study showed that several multivalent galactose-specific lectins including the 14-kDa lectin from calf spleen and the lectins from Erythrina indica, Erythrina cristagalli, and soybean agglutinin formed specific cross-linked complexes with the glycoprotein asialofetuin (ASF) [Mandal, D. K., & Brewer, C. F. (1992) Biochemistry 31, 8465-8472]. In the present study, we have used quantitative precipitation analysis to compare the cross-linking activities of the Gal/GalNAc-specific lectin from Erythrina corallodendron (ECorL) and the recombinant protein (rECorL) which lacks the covalently linked heptasaccharide chains of the native lectin, with ASF. At low concentrations of ASF relative to the lectin, native dimeric ECorL binds to each of the three terminal Gal residues of the three N-linked triantennary chains of ASF and precipitates as a cross-linked complex at a ratio of 1:9 ASF/lectin (monomer). With increasing concentrations of ASF, the 1:9 complex changes to a 1:3 ASF/lectin complex, and at higher ASF concentrations, a 1:1 cross-linked complex forms. However, rECorL, which possesses the same specificity and binding affinity as the native lectin, forms only the 1:9 and 1:3 ASF/lectin complexes. Other Erythrina lectins examined, all of which have covalently attached carbohydrate and are structurally similar to ECorL, show the same cross-linking behavior as native ECorL. On the other hand, the dimeric 14-kDa calf spleen lectin which lacks covalently attached carbohydrate forms only 1:9 and 1:3 cross-linked complexes with ASF [Mandal, D. K., & Brewer, C. F. (1992) Biochemistry 31, 8465-8472].(ABSTRACT TRUNCATED AT 250 WORDS)

Asialoglycoproteins↗

PubMed1994

Determination of the concentrations of oligosaccharides, complex type carbohydrates, and glycoproteins using the phenol-sulfuric acid method.

The concentrations of methyl glycosides, oligosaccharides, glycopeptides, and glycoproteins can be accurately determined by using calibration curves composed of the appropriate monosaccharide(s) obtained with a modified version of the colorimetric phenol-sulfuric acid method. Calibration curves of micrograms sugar vs. 490 nm for Man, Glc, or Gal are shown to provide reliable determinations (typically +/- 3-4%) of corresponding methyl glycosides and linear and branched-chain oligosaccharides containing the corresponding reactive hexose residue. For complex oligosaccharides containing a known mixture of reactive hexose units, the appropriate mixture of monosaccharides are shown to provide equally accurate calibration curves for concentration determinations. In the case of the soybean agglutinin, which is a tetramer possessing one Man9 oligomannose-type chain per subunit, the protein concentration was determined from the Man calibration curve which agreed with that obtained from the molar extinction coefficient of the protein.

Carbohydrate Conformation↗

PubMed1994

Thermodynamics of lectin-carbohydrate interactions. Titration microcalorimetry measurements of the binding of N-linked carbohydrates and ovalbumin to concanavalin A.

The thermodynamics of binding of concanavalin A (Con A) with a series of linear and branched chain oligosaccharides including certain N-linked complex type and oligomannose type carbohydrates and a fraction of quail ovalbumin containing Man7 and Man8 oligomannose chains have been determined using titration microcalorimetry. Methyl3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, a branch chain trisaccharide moiety found in all N-linked carbohydrates which possesses approximately 60-fold higher affinity than methyl alpha-D-mannopyranoside, exhibited a change in enthalpy of binding (delta H) of -14.4 kcal mol-1 as compared to -8.2 kcal mol-1 for the monosaccharide. This demonstrates that Con A possesses an extended binding site for the trimannoside. However, a biantennary complex type carbohydrate with terminal beta (1,2)-GlcNAc residues which binds with 3-fold higher affinity than the trimannoside possesses a delta H of only -10.6 kcal mol-1. A plot of -delta H versus -T delta S for the carbohydrates in the present study showed positive deviations in -T delta S for the complex type carbohydrate, as well as alpha (1,2)-di- and trimannosyl oligosaccharides which are part of the structures of oligomannose type carbohydrates. The relative favorable changes in binding entropies of these compounds are attributed to the presence of multiple internal and terminal residues in each molecule which can independently bind to the monosaccharide binding site of the lectin. The delta H values for the complex type carbohydrate and the alpha (1,2) mannose oligosaccharides were also approximately -2.5 kcal mol-1 greater than that of methyl alpha-D-mannopyranoside, indicating some extended binding site interactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Studies of the binding specificity of concanavalin A. Nature of the extended binding site for asparagine-linked carbohydrates.

In the preceding paper [Mandal, D. K., Kishore, N., & Brewer, C. F. (1994) Biochemistry (preceding paper in this issue)] the trisaccharide 3,6-di-O-(alpha-D-mannopyranosyl)-D-mannose, which is present in all asparagine-linked carbohydrates, was shown by titration microcalorimetry to bind to the lectin concanavalin A (Con A) with nearly -6 kcal mol-1 greater enthalpy change (delta H) than methyl alpha-D-mannopyranoside (Me alpha Man). These results indicate that Con A possesses an extended binding site for the trisaccharide. In the present paper, we have investigated the binding of a series of synthetic analogs of the methyl alpha-anomer of the trisaccharide using hemagglutination inhibition, solvent proton magnetic relaxation dispersion (NMRD), near ultraviolet circular dichroism, and titration microcalorimetry measurements. Four of the analogs tested possess an alpha-glucosyl or alpha-galactosyl residue substituted at either the alpha(1-6) or alpha(1-3) position. Analysis of the data indicates that the alpha(1-6) residue of the parent trimannoside binds to the so-called monosaccharide site and the alpha(1-3) residue to a weaker secondary site. Binding at the secondary site involves unfavorable interactions of the 2-equatorial hydroxyl of the alpha(1-3) Glc derivative since this analog binds with 12-fold lower affinity and -3.4 kcal mol-1 lesser delta H than the trimannoside, whereas the alpha(1-3)-2-deoxyGlc analog possesses essentially the same affinity and delta H as the trimannoside.(ABSTRACT TRUNCATED AT 250 WORDS)

Asparagine↗

PubMed1994

Investigation of protein-protein noncovalent interactions in soybean agglutinin by electrospray ionization time-of-flight mass spectrometry.

Noncovalent interactions in soybean agglutinin (SBA) were studied on an electrospray ionization (ESI) time-of-flight mass spectrometer constructed recently at the University of Manitoba. The high m/z range and high sensitivity of the instrument together with mild ESI interface conditions turned out to be ideal for detecting this noncovalently bonded tetrameric protein (MW approximately 116,000 Da) in low charge states (z = 23 to 27). By altering the acetonitrile content of the SBA solutions it was shown that the observed SBA tetramers are due to structurally specific noncovalent associations in solution. Octamers and dodecamers (MW approximately 350,000 Da) were also detected. Information on the quaternary structure of the tetramers was obtained by analyzing the fragment-ion spectrum resulting from the collision-induced dissociation of the tetramer ions.

Lectins↗

PubMed1993

Selective inhibition of N-acetylglucosamine and galactose-specific lectins including the 14-kDa vertebrate lectin by novel synthetic biantennary oligosaccharides.

A novel series of synthetic biantennary tri-, penta- and hepta-saccharides with terminal beta-GlcNAc, beta-LacNAc and alpha NeuAc(2,6)beta LacNAc residues, respectively, [LacNAc = Gal beta (1,4)Glc-NAc] connected to a core Gal residue were evaluated for their inhibitory potencies for specific plant and animal lectins. Six isomeric trisaccharides with two beta-GlcNAc residues at the 2,3-, 2,4-, 2,6-, 3,4-, 3,6-, or 4,6-positions of the core Gal were tested for their hemagglutination inhibition activities against two GlcNAc-specific lectins, Griffonia simplicifolia II (GS II) and wheat germ agglutinin (WGA). The 2,3-, 2,4-, 2,6- and 3,6-trisaccharides inhibited WGA 12-50 times more strongly than GlcNAc, whereas only weak or no inhibition was observed with GS II. The 3,4- and 4,6-trisaccharides did not inhibit either of the lectins. Six biantennary isomeric pentasaccharides containing two terminal beta-LacNAc residues with branching patterns similar to the trisaccharides showed selective hemagglutination inhibition of five Gal/GalNAc-specific plant lectins and the 14-kDa Gal-specific calf spleen lectin. The plant lectins include the soybean agglutinin (SBA), ricin agglutinin-I (RCA-I), and three Erythrina lectins with similar specificities: Erythrina indica (EIL), E. corallodendron (ECorL), and E. cristagalli (ECL). The 2,3-pentasaccharide inhibited only SBA and the 14-kDa lectin, and thus was a selective inhibitor among the plant lectins. The 2,6-pentasaccharide inhibited SBA, ECL and the 14-kDa lectin, but not RCA-I or the two other Erythrina lectins. The 4,6-pentasaccharide did not inhibit any of the plant lectins, but was a specific inhibitor of the 14-kDa calf spleen lectin. Synthetic heptasaccharides analogs with 2,4-, 2,6-, 3,6- and 4,6-branching patterns and terminal alpha(2,6)NeuAc residues all showed 25-fold stronger inhibition against the alpha(2,6)sialic-acid-specific elderberry (Sambucus nigra L.) bark lectin as compared to a monovalent disaccharide alpha NeuAc(2,6)beta GalOR. The lack of inhibition of alpha NeuAc(2,6)beta GalOR derivatives methylated at the C6 of the Gal residue and a sulfur-linked thiosialoside derivative demonstrates that the 2,6-anomeric linkage region is important for lectin recognition. Selective inhibition of the Gal/GalNAc-specific lectins was observed for two isomeric C6 methyl-substituted Gal derivatives of methyl beta-LacNAc which possess different preferred rotamer orientations about the C5-C6 bond of the Gal residue.

Acetylglucosamine↗

PubMed1993

Differences in the binding affinities of dimeric concanavalin A (including acetyl and succinyl derivatives) and tetrameric concanavalin A with large oligomannose-type glycopeptides.

Dimeric derivatives of concanavalin A (Con A) such as acetyl- and succinyl-Con A have been used for years as probes of cellular membranes. The altered binding and biological activities of these derivatives relative to native tetrameric Con A have generally been attributed to their reduced valence. However, the present study shows that acetyl- and succinyl-Con A possess lower affinities than tetrameric Con A toward certain oligomannose-type glycopeptides which are found on the surface of cells. It has previously been shown that native tetrameric Con A possesses 5-30-fold enhanced affinities toward Man7-Man9 oligomannose-type glycopeptides, respectively, relative to Man5 and Man6 oligomannose-type glycopeptides [Bhattacharyya, L., & Brewer, C. F. (1989) Eur. J. Biochem. 178, 721-726]. Using titration microcalorimetry and hemagglutination inhibition measurements, methyl alpha-D-mannopyranoside, methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside (which binds with about 60-fold higher affinity than methyl alpha-D-mannopyranoside and is the major Con A binding epitope on oligomannose-type carbohydrates), and a Man5 oligomannose-type oligosaccharide are shown to bind to underivatized dimeric Con A at pH 5.2 and acetyl- and succinyl-Con A at pH 7.2 with affinities equal to those of native tetrameric Con A. However, a mixture of Man7 and Man8 glycopeptides and a Man9 oligomannose-type glycopeptide were shown to bind to underivatized dimeric Con A and acetyl- and succinyl-Con A with affinities only about 2-fold higher than the Man5 oligosaccharide, in contrast to the higher affinities of native tetrameric Con A for these carbohydrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed1992

Interactions of concanavalin A with glycoproteins: formation of homogeneous glycoprotein-lectin cross-linked complexes in mixed precipitation systems.

We have previously demonstrated that the interactions between branched chain oligosaccharides and glycopeptides isolated from glycoproteins and glycolipids with specific lectins lead to the formation of homopolymeric carbohydrate-protein cross-linked complexes, even in the presence of mixtures of the carbohydrates or lectins [cf. Bhattacharyya, L., Fant, J., Lonn, H., & Brewer, C. F. (1990) Biochemistry 29, 7523-7530]. Recently, we have shown that highly ordered cross-linked lattices are formed between the tetrameric glycoprotein soybean agglutinin (SBA), which possesses a Man9 oligomannose chain per monomer, and the Glc/Man-specific plant lectin concanavalin A (Con A) [Khan, M. I., Mandal, D. K., & Brewer, C. F. (1991) Carbohydr. Res. 213, 69-77]. Using radiolabeling and quantitative precipitation techniques, we show in the present study that Con A binds and forms unique cross-linked complexes with four different glycoproteins having different numbers and types of carbohydrate chains as well as different quaternary structures. The glycoproteins include quail ovalbumin, Lotus tetragonolobus isolectin A (LTL-A), Erythrina cristagalli lectin (ECL), and Erythrina corallodendron lectin (EcorL). The results show that a preparation of quail ovalbumin containing either one Man7 or Man8 oligomannose chain per molecule forms a 1:2 cross-linked complex with tetrameric Con A, thereby demonstrating bivalency of the single carbohydrate chain(s) on the glycoprotein. Tetrameric LTL-A and dimeric ECL, which possess two xylose-containing carbohydrate chains per monomer, both form 1:2 and 1:1 cross-linked complexes (per monomer) of glycoprotein to lectin, depending on their relative ratios in solution. However, dimeric EcorL, which has the same carbohydrate structure and number of chains as ECL, forms only a 1:2 cross-linked complex with tetrameric Con A.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Cross-linking activity of the 14-kilodalton beta-galactoside-specific vertebrate lectin with asialofetuin: comparison with several galactose-specific plant lectins.

We have previously shown that plant lectins with a wide range of carbohydrate binding specificities can bind and cross-link (precipitate) specific multiantennary oligosaccharides and glycopeptides [cf. Bhattacharyya, L., Fant, J., Lonn, H., & Brewer, C. F. (1990) Biochemistry 29, 7523-7530]. This leads to a new source of binding specificity: namely, the formation of homogeneous cross-linked lattices between lectins and carbohydrates. Recently, we have demonstrated the existence of highly ordered cross-linked lattices that form between the D-Man/D-Glc-specific plant lectin concanavalin A and the soybean agglutinin which is a tetrameric glycoprotein possessing a single Man9 oligomannose chain per monomer [Khan, M. I., Mandal, D. K., & Brewer, C. F. (1991) Carbohydr. Res. 213, 69-77]. In the present study, we have compared the ability of the 14-kDa beta-galactoside-specific lectin from calf spleen, a dimeric S-type animal lectin, and several galactose-specific plant lectins from Erythrina indica, Erythrina cristagalli, and Glycine max (soybean agglutinin) to form specific cross-linked complexes with asialofetuin (ASF), a 48-kDa monomeric glycoprotein, using quantitative precipitation analyses. The results show the formation of 1:9 and 1:3 stoichiometric cross-linked complexes (per monomer) of ASF to the 14-kDa lectin, depending on their relative ratio in solution. Evidence indicates that the three triantennary N-linked complex-type oligosaccharide chains of ASF mediate the cross-linking interactions and that each chain expresses either trivalency in the 1:9 cross-linked complex or univalency in the 1:3 complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Formation of homogeneous carbohydrate-lectin cross-linked precipitates from mixtures of D-galactose/N-acetyl-D-galactosamine-specific lectins and multiantennary galactosyl carbohydrates.

Quantitative precipitation studies have shown that the Man/Glc-specific lectin concanavalin A (ConA) forms homogeneous (homopolymeric) cross-linked precipitates with individual asparagine-linked oligomannose and bisected hybrid-type glycopeptides in the presence of binary mixtures of the carbohydrates [Bhattacharyya, L., Khan, M. I. & Brewer, C. F. (1988) Biochemistry 27, 8762-8767]. The results indicate that the ConA-glycopeptide precipitates are highly organized cross-linked lattices that are unique for each carbohydrate. Using similar techniques, the present study shows that the Gal-specific lectins from Erythrina indica and Ricinus communis (agglutinin I) form homogeneous cross-linked complexes with individual carbohydrates in binary mixtures of triantennary and tetraantennary complex-type oligosaccharides with terminal Gal residues. Conversely, binary mixtures of Gal/GalNAc-specific lectins from E. indica, Erythrina cristagalli, Erythrina flabelliformis, R. communis, soybean (Glycine max), and Wistaria floribunda (tetramer) in the presence of a naturally occurring or synthetic branched-chain oligosaccharide with terminal GalNAc or Gal residues provide evidence for the formation of separate cross-linked lattices between each lectin and the carbohydrate. The present results therefore demonstrate the formation of homogeneous lectin-carbohydrate cross-linked lattices in (a) a mixture of branched-chain complex-type oligosaccharides in the presence of a specific Gal/GalNAc-binding lectin, and (b) a mixture of lectins with similar physicochemical and carbohydrate binding properties in the presence of an oligosaccharide. These findings show that lectin-carbohydrate cross-linking interactions provide a high degree of specificity which may be relevant to their biological functions as receptors.

Acetylgalactosamine↗

PubMed1992

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.

Carbohydrate Conformation↗

PubMed1991

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.

Catalysis↗

PubMed1991

Interactions of concanavalin A with glycoproteins. A quantitative precipitation study of concanavalin A with the soybean agglutinin.

Certain oligomannose-type glycopeptides have been previously shown to be bivalent for binding to concanavalin A and capable of precipitating the lectin by forming homogeneous cross-linked lattices [L. Bhattacharyya, M. I. Khan, and C.F. Brewer, Biochemistry, 27 (1988) 8762-8767]. In the present study, the effect of protein environment on the binding properties of an oligomannose-type oligosaccharide has been examined through quantitative precipitation analysis of the interactions of concanavalin A (Con A) with the soybean (Glycine max) agglutinin (SBA), which is a tetrameric glycoprotein possessing a single Man9-oligomannose chain per monomer. The results showed that SBA forms two different types of cross-linked complexes with tetrameric Con A, depending on the relative ratio of the two molecules in solution. At a concentration of one equivalent or less, SBA forms a 1:1 complex with Con A. At concentrations exceeding one equivalent, SBA forms a 2:1 complex with Con A. However, SBA forms only 1:1 cross-linked complexes with dimeric forms of Con A, such as acetyl- and succinyl-Con A. The results demonstrated that the total valency of the carbohydrate of SBA is a function of both the quaternary structure of Con A, as well as the relative ratio of SBA to Con A. In addition, the individual Man9-oligosaccharide, which as a glycopeptide is bivalent for binding to Con A, expresses univalency when present on the protein matrix of SBA.

Carbohydrate Sequence↗

PubMed1991

Interactions of asparagine-linked carbohydrates with concanavalin A. Nuclear magnetic relaxation dispersion and circular dichroism studies.

By using near-UV circular dichroism (CD) and solvent proton nuclear magnetic relaxation dispersion measurements, three different conformational states have been detected in Ca(2+)-Mn(2+)-concanavalin A upon binding a variety of asparagine-linked carbohydrates. Two of these transitions have been described previously, one for the binding of monosaccharides such as methyl alpha-D-mannopyranoside and oligosaccharides with terminal alpha-Glc or alpha-Man residues, and the second for the binding of oligomannose and complex type carbohydrates (Brewer, C. F., and Bhattacharyya, L. (1986) J. Biol. Chem. 261, 7306-7310). The third transition occurs upon binding a bisected biantennary complex type carbohydrate with terminal GlcNAc residues. Temperature-dependent nuclear magnetic relaxation dispersion and CD measurements have identified regions of the protein near the two metal ion binding sites that are associated with the conformation changes, and Tyr-12, which is part of the monosaccharide binding site, as responsible for the CD changes. The results support our previous conclusions that the rotamer conformation of the (alpha 1,6) arm of bisected complex type oligosaccharides binds to concanavalin A with dihedral angle omega = -60 degrees whereas nonbisected complex type oligosaccharides bind with omega = 180 degrees (Bhattacharyya, L., Haraldsson, M., and Brewer, C. F. (1987) J. Biol. Chem. 262, 1294-1299). The present findings also explain the effects of increasing chain length of bisected complex type carbohydrates on their interactions with the lectin.

Asparagine↗

PubMed1990

Substrate-induced activation of maltose phosphorylase: interaction with the anomeric hydroxyl group of alpha-maltose and alpha-D-glucose controls the enzyme's glucosyltransferase activity.

Maltose phosphorylase, long considered strictly specific for beta-D-glucopyranosyl phosphate (beta-D-glucose 1-P), was found to catalyze the reaction beta-D-glucosyl fluoride + alpha-D-glucose----alpha-maltose + HF, at a rapid rate, V = 11.2 +/- 1.2 mumol/(min.mg), and K = 13.1 +/- 4.4 mM with alpha-D-glucose saturating, at 0 degrees C. This reaction is analogous to the synthesis of maltose from beta-D-glucose 1-P + D-glucose (the reverse of maltose phosphorolysis). In acting upon beta-D-glucosyl fluoride, maltose phosphorylase was found to use alpha-D-glucose as a cosubstrate but not beta-D-glucose or other close analogs (e.g., alpha-D-glucosyl fluoride) lacking an axial 1-OH group. Similarly, the enzyme was shown to use alpha-maltose as a substrate but not beta-maltose or close analogs (e.g., alpha-maltosyl fluoride) lacking an axial 1-OH group. These results indicate that interaction of the axial 1-OH group of the disaccharide donor or sugar acceptor with a particular protein group near the reaction center is required for effective catalysis. This interaction appears to be the means that leads maltose phosphorylase to promote a narrowly defined set of glucosyl transfer reactions with little hydrolysis, in contrast to other glycosylases that catalyze both hydrolytic and nonhydrolytic reactions.

Binding Sites↗