Chemical mapping of the active site of the glucoamylase ofAspergillus niger

Abstract

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...

Automated docking of monosaccharide substrates and analogues and methyl α-Acarviosinide in the glucoamylase active site

“…This is analogous to the conformation found in studies of GA complexed with different inhibitors, 6-11 most structures making a hydrogen bond with Asp55 as maltosides do. 15,50 Both gt and tg conformations of the hydroxymethyl group are also possible and often lead to distinct clusters of docked structures, but they cause a molecular interaction energy penalty. The OH-6 A group is not only essential for hydrolysis of maltose and isomaltose, [50][51][52] but is equally necessary for the condensation of Dglucose and its deoxy derivatives.…”

“…15,50 Both gt and tg conformations of the hydroxymethyl group are also possible and often lead to distinct clusters of docked structures, but they cause a molecular interaction energy penalty. The OH-6 A group is not only essential for hydrolysis of maltose and isomaltose, [50][51][52] but is equally necessary for the condensation of Dglucose and its deoxy derivatives. 3,53 The positional flexibility of this hydroxyl group in the GA active site could have some mechanistic implications both in substrate binding and in catalysis.…”

“…On the other hand, there is only a small interaction with OH-2 B , which forms a weak hydrogen bond with Glu180 in the GA-acarbose complex 9,11 and the GAmaltose transition complex. 15,50,59 CONCLUSIONS Favorable docking between monosaccharide substrates and monomeric and dimeric inhibitors, especially with protonated inhibitors, has been simulated by Monte Carlo techniques. Especially noteworthy is the correspondence with numerous crystallographic results, which suggest that tight binding leading to strong inhibition is obtained by charged forms forming saltlike links with Glu179.…”

Automated docking of glucosyl disaccharides in the glucoamylase active site

“…X100 GA catalytic domain obtained by X-ray crystallography in its native state or complexed with different inhibitors [8][9][10][11][12][13] was the basis for modeling the approximate interaction of GA with the substrates methyl a-maltoside and methyl a-isomaltoside in the first and second subsites. 14,15 The main interactions of these substrates with active-site residues and the catalytic water molecule were identified, with most of these residues being conserved in fungal, yeast, and bacterial GAs. 14,16,17 Automated docking with AutoDock, combining Monte Carlo methods with simulated annealing, has allowed the study of the interaction of proteins with small flexible molecules [18][19][20] and with other proteins.…”

Mutational analysis of Aspergillus awamori glucoamylase selectivity to improve glucose yield

“…niger GA activity ; Trp590 and Trp615 are responsible for starch granule adsorption (Svensson et al, 1986b). Studies with substrate analogues showed that the 3-, 4'-, and 6'-0H groups of maltosides as well as the 4-, 4'-, and 6'-0H groups of isomaltosides are required for rapid hydrolysis Pedersen, 1987, 1988;Bock et al, 1991;Lemieux et al, 1996).…”