. Can. J. Chem. 63, 2644Chem. 63, (1985. Using a radioimmunoassay to measure the relative potencies as inhibitors of a wide number of chemically modified structures related to the Lewis b human blood group determinant, it was found that derivatives of the Lewis b (a~Fuc(l+2)p~Gal(l+ 3)[a~Fuc(I+4)]P~GlcNAc) and the Y (a~Fuc(l+2)~~Gal(l+4)[cr~Fuc(1+3)]f3~GlcNAc) determinants are complexed by the lectin IV of Grzffonia simplicifolia through the recognition of a topographical feature that is common to both the tetrasaccharides. This surface provides a nonpolar region formed by the two methyl groups of the fucose units and extends along the C-1-0-5-C-5 side of the a~Fuc(l+2) unit and terminates at one end by a polar grouping which is formed by OH-3 and OH-4 of the PDGal unit and OH-4 of the a~Fuc(1-4) unit. Association constants were determined from changes in ultraviolet absorption that occur as the result of complex formation. For the reaction of the Lewis b-0CH3 tetrasaccharide, the thermodynamic parameters were found to be AH = -13 kcal/mol and AS = -22 cal/mol/K. The inhibition data for the relevant monodeoxy derivatives indicated that OH-2 and OH-3 of both of the a~F u c units are not directly involved in the binding reaction. The basis for drawing these conclusions was strengthened by finding that the reaction of the simple Lewis
. Can. J. Chem. 68, 1116 (1990).The lectin IV of Griffonia simplicifolia (GS4) specifically binds the terminal tetrasaccharide unit of the Lewis b human blood group determinant (~e~) .The single crystal X-ray analysis of the complex with L~~-o M~ has demonstrated that the binding site on the lectin is a shallow depression with a negatively charged aspartate side chain at the bottom of the cavity. In addition to this aspartate, a serine and an asparagine side chain provide the polar groups that hydrogen bond to the three hydroxyl groups of Leb, which has been termed the key polar grouping for complex formation. A notable characteristic of the binding site is that five aromatic amino acid side chains (one Phe, two Tyr, and two Trp residues) surround these polar interactions and make van der Waals contacts with the tetrasaccharide. Thus, as predicted from previous solution binding studies, extensive nonpolar interactions are involved, which contribute importantly both to the specificity of the reaction and the stability of the noncovalent complex that is formed. These results represent the first structural example of the molecular recognition of a human blood group determinant by the receptor site of a protein. Extensive sequence homology exists between GS4 and the concanavalin A (Con A), pea, and favin lectins. The main hydrophilic groups of the carbohydrate-binding site of GS4 and Con A are aspartate, asparagine, and serine residues; the homology suggests that the serine is replaced by asparagine in the case of the pea and favin lectins. It appears probable that these two latter lectins possess very similar, if not identical, specificities.Key words: lectin, carbohydrate, molecular recognition, binding.Lours T. J. DELBAERE, MARGARET VANDONSELAAR, LATA PRASAD, J. WILSON QUAIL, JOYCE R. PEARLSTONE, MICHAEL R. CARPENTER, LAWRENCE B. SMILLIE, PANDURANG V. NIKRAD, ULRIKE SPOHR et RAYMOND U. LEMIEUX. Can. J. Chem. 68, 1116 (1990).La lectine IV du Grrffonia simplicifolia (GS4) se lie sptcifiquement a l'unitt tktrasaccharide terminale du determinant du groupe sanguin humain Lewis b (Leb). Une analyse par diffraction des rayons-X effectute sur un cristal unique de son complexe avec le Leb-OM^ a perrnis de dtmontrer que le site de liaison de la lectine est une ltgtre dkpression au fond de laquelle on retrouve une chaine lattrale d'aspartate chargke nkgativement. En plus de cet aspartate, des chaines lattrales de strine et d'asparagine fournissent des groupements polaires qui foment des points hydrogenes avec les trois groupements hydroxyles du ~e~; il s'agit du groupement polaire clt pour la formation de complexes. Le fait que cinq chaines lattrales formtes d'acides amints arornatiques entourent ces interactions dipolaires et qu'elles ttablissent des contacts de van der Waals avec le tktrasaccharide est une caracteristique notable de ce site de liaison. Tel que prkdit 2 partir d'ttudes anttrieures de liaisons en solution, il existe donc de nornbreuses interactions non-polaires qui apportent une contribution importante tant...
The chemical mapping of the regions of H-type 2 human blood group-related trisaccharide (Fuc alpha (1-2)Gal beta (1-4)GlcNAc beta Me) that are recognized by three different lectins, the so-called epitopes, are reviewed together with an account of how and why oligosaccharides form specific complexes with proteins as presently viewed in this laboratory. The occasion is used to report the synthesis of the various mono-O-methyl derivatives of the above trisaccharide that were used in these investigations. Also, Fuc alpha (1-2)Gal beta (1-4)Xyl beta Me was synthesized in order to examine whether or not the hydroxymethyl group of the GlcNAc residue participates in the binding reaction.
ULRIKE SPOHR, EUGENIA PASZKIEWICZ-HNATIW, NAOHIKO MORISHIMA, and RAYMOND U. LEMIEUX. Can. J . Chem. 70, 254 (1992).The relative potencies of a wide variety of deoxygenated derivatives of the methyl glycoside of a-L-Fuc-(1 -+ 2)-P-D-Gal-(1 -+ 4)-p-~-GlcNAc (the H-type 2 human blood group related trisaccharide) for the inhibition of the binding of an artificial H-type 2 antigen by the lectin I of Ulex europaeus confirmed the previous evidence that the key and productive interaction involves only the three hydroxyl groups of the a-L-fucose unit, the hydroxyl at the 3-position of the P-D-galactose residue, and the nonpolar groups in their immediate environment. Except for the acetamido group and the hydroxymethyl of the P -D -G~~ unit, which stay in the aqueous phase, on complex formation the remaining three hydroxyl groups appear to come to reside at or near the periphery of the combining site since their replacement by hydrogen causes relatively small changes (<* 1 kcal/mol) in the stability of the complex (AGO). Relatively much larger but compensating changes occur for the enthalpy and entropy terms, and these may arise primarily from the differences in the water structure about the periphery of the combining site and the oligosaccharide both prior to and after complexation. It is proposed that steric constraints lead to an ordered state of the water molecules hydrogen-bonded to the polar groups within the cleft formed by the key region of the amphiphilic combining site. Their release to form less ordered clusters of more strongly hydrogen-bonded water molecules in bulk solution would contribute importantly to the driving force for complexation. It is demonstrated that the surface used for the binding of H-type 2-OMe by a monoclonal anti-H antibody is virtually identical to that used by the Ulex lectin.Key words: molecular recognition, H-type 2 blood group determinant and deoxygenated derivatives, lectin I of Ulex europaeus, anti-H-type 2 monoclonal antibody, enthalpy-entropy compensation. Les efficacitCs relatives d'un grand nombre de dCrivCs dCsoxygCnCs du glycoside de mCthyle de l'a-L-Fuc-(I -+ 2)-P-D-Gal-(I -+ 4)-P-D-GlcNAc (trisaccharide reliC au groupe sanguin H de type 2 de l'homme) comme inhibiteurs de la fixation d'un antigkne artificiel du H de type 2 par la lectine I de 1'Ule.x europaeus ont confirmi les donnCes antCrieures suggCrant que cette interaction importante et productive n'implique que les trois groupes hydroxyles de I'unitC a-L-fucose, le groupe hydroxyle.en position 3 du rCsidu P-D-galactose et les groupes non-polaires qui se trouvent dans leur environnement immCdiat. A l'exception du groupe acetamido et de I'hydroxymCthyle de I'unitC P-D-Gal qui restent dans la phase aqueuse lors de la formation du complexe, il semble que les trois autres groupes hydroxyles se retrouvent a, ou pres de, la pCriphCrie du site qui effectue la combinaison; en effet, leur remplacement par de l'hydrogkne ne provoque que de faibles changements (<* 1 kcal/mol) dans la stabilitk du complexe (AGO). Des changements r...
Abstract:A recently developed technique for the probing of the combining sites of lectins and antibodies, to establish the structure of the epitope that is involved in the binding of an oligosaccharide, is used to study the binding of methyl aisomaltoside by the enzyme glucoamylase. The procedure involved the determination of the effects on the kinetics of hydrolysis of both monodeoxygenation and mono-0-methylation at each of the seven hydroxyl groups in order to gain an estimate of the differential changes in the free energies of activation, AAG' . As expected, from previous publications, both deoxygenation and 0-methylation of OH-4 (reducing unit), OH-4', or OH-6' strongly hindered hydrolysis, whereas the kinetics were virtually unaffected by either the substitutions at OH-2 or structural changes at C-I. The substitutions at OH-3 caused increases of 2.1 and 1.9 kcallmol in the AAG'. In contrast, whereas deoxygenation of either OH-2' or OH-3' caused much smaller (0.96 and 0.52 kcallmol) increases in AAG', the mono-0-methylations resulted in severe steric hindrance to the formation of the activated complex. The relatively weak effects of deoxygenation suggest that the hydroxyl groups are replaced by water molecules and thereby participate in the binding by contributing effective complementarity. Methyl a-isomaltoside was docked into the combining site of the X-ray crystal structure at 2.4 A resolution of the complex with the inhibitor acarbose. A fit free of steric interactions with the protein was found that has the methyl a-glucopyranoside unit in the normal "C, conformation and the other glucose unit approachin a half-chair conformation with the interunit fragment defined by the torsion angles +IIJJ/W = 74°/1340/ % 166' (0-5'-C-l'h-64-6%-5-0-5).The model provides a network of hydrogen bonds that appears to well represent the activated complex formed by the glucoamylase with both maltose and isomaltose since the structures appear to provide a sound rationale for both the specificity and catalysis provided by the enzyme.Key war-ds: monodeoxy and mono-0-methyl derivatives of methyl a-isomaltoside, enzyme binding domain, functioning of glucoamylase, differential changes in free energy of activation, characteristics of hydrogen bonding networks. Resum6: Dans le but d'Ctudier la fixation de l'a-isomaltoside de mCthyle par l'enzyme glucoamylase, on a fait appel B une technique, dCveloppCe rCcemment pour I'examen des sites de fixation des lectines et des anticorps, pour Ctablir la structure de I'Cpitope impliqui dans la fixation d'un oligosaccharide. I,a procCdure implique la dktermination des effets, provoquCs sur la cinCtique d'hydrolyse, par la monodCsoxygCnation ainsi que la mono-0-methylation de chacun des sept groupes hydroxyles; on espitre ainsi obtenir une evaluation des changements diffkrentiels dans les Cnergies libres d'activation, AAG'. Tel qu'on peut s'y attendre sur la base des publications antkrieures, tant la dCsoxygCnation que la 0-mtthylation des groupes OH-4 (unit6 rkductrice), OH-4' ou OH-6' empsc...
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