Roles of the aromatic side chains in the binding of substrates, inhibitors, and cyclomalto-oligosaccharides to the glucoamylase from Aspergillus niger probed by perturbation difference spectroscopy, chemical modification, and mutagenesis

Birte Svensson, Michael Sierks

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70 Citations (Scopus)

Abstract

The roles of the aromatic side chains of the glucoamylase from Aspergillus niger in the binding of ligands, as determined by differences spectroscopy using four types of inhibitors (a) valienamine-derived, (b) 1-deoxynojrimyscins, (c) d-glucono-1,5-lactone, and (d), two types of disaccharide substrates (a) α-(1»4)-linked and (b) α-(1»6)-linked, and three cyclomalto-oligosaccharides (cyclodextrins, CDs) are discussed. An unusual change in absorbance from 300 to 310-320 nm, obtained only with valienamine-derived inhibitors or when d-glucono-1,5-lactone and maltose are combined, is concluded to arise when subsite 2 is occupied in a transition-state-type of complex. The single mutations of two residues thought to be involved in binding, namely, Tyr116»Ala and Trp120»Phe, alter, but do not abolish this perturbation. The perturbations in the spectra also suggest that maltose and isomaltose have different modes of binding. The following Kd values (m) were determined: acarbose, < x 10-12; methyl acarviosinide, 1.6 x 10-6; and the d-gluco and l-ido forms of hydrogenated acarbose, 1.4 x 10-8 and 5.2 x 10-6, respectively. Therefore, both the valienamine moiety and the chain length of acarbose are important for tight binding. In contrast to the valienamine-derived inhibitors, none of the 1-deoxynojirimycin type protected glucoamylase against inactivating oxidation of tryptophyanyl residues, although each had a Kd value of ∼ 4 x 10-6m. There are two distinct carbohydrate-binding areas in glucoamylase, namely, the active site in the catalytic domain and a strach-granule-binding site in the C-terminal domain. The α-,β-, and γ-CDs have high affinity for the starch-binding domain and low affinity for the active site, whereas the reverse was found for acarbose.

Original languageEnglish (US)
Pages (from-to)29-44
Number of pages16
JournalCarbohydrate Research
Volume227
Issue numberC
DOIs
StatePublished - Apr 6 1992
Externally publishedYes

Fingerprint

Acarbose
Glucan 1,4-alpha-Glucosidase
Mutagenesis
Aspergillus niger
Aspergillus
Chemical modification
Oligosaccharides
Spectrum Analysis
Spectroscopy
Catalytic Domain
Maltose
Cyclodextrins
Lactones
Substrates
Isomaltose
1-Deoxynojirimycin
Disaccharides
Chain length
Starch
Binding Sites

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Organic Chemistry

Cite this

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title = "Roles of the aromatic side chains in the binding of substrates, inhibitors, and cyclomalto-oligosaccharides to the glucoamylase from Aspergillus niger probed by perturbation difference spectroscopy, chemical modification, and mutagenesis",
abstract = "The roles of the aromatic side chains of the glucoamylase from Aspergillus niger in the binding of ligands, as determined by differences spectroscopy using four types of inhibitors (a) valienamine-derived, (b) 1-deoxynojrimyscins, (c) d-glucono-1,5-lactone, and (d), two types of disaccharide substrates (a) α-(1»4)-linked and (b) α-(1»6)-linked, and three cyclomalto-oligosaccharides (cyclodextrins, CDs) are discussed. An unusual change in absorbance from 300 to 310-320 nm, obtained only with valienamine-derived inhibitors or when d-glucono-1,5-lactone and maltose are combined, is concluded to arise when subsite 2 is occupied in a transition-state-type of complex. The single mutations of two residues thought to be involved in binding, namely, Tyr116»Ala and Trp120»Phe, alter, but do not abolish this perturbation. The perturbations in the spectra also suggest that maltose and isomaltose have different modes of binding. The following Kd values (m) were determined: acarbose, < x 10-12; methyl acarviosinide, 1.6 x 10-6; and the d-gluco and l-ido forms of hydrogenated acarbose, 1.4 x 10-8 and 5.2 x 10-6, respectively. Therefore, both the valienamine moiety and the chain length of acarbose are important for tight binding. In contrast to the valienamine-derived inhibitors, none of the 1-deoxynojirimycin type protected glucoamylase against inactivating oxidation of tryptophyanyl residues, although each had a Kd value of ∼ 4 x 10-6m. There are two distinct carbohydrate-binding areas in glucoamylase, namely, the active site in the catalytic domain and a strach-granule-binding site in the C-terminal domain. The α-,β-, and γ-CDs have high affinity for the starch-binding domain and low affinity for the active site, whereas the reverse was found for acarbose.",
author = "Birte Svensson and Michael Sierks",
year = "1992",
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T1 - Roles of the aromatic side chains in the binding of substrates, inhibitors, and cyclomalto-oligosaccharides to the glucoamylase from Aspergillus niger probed by perturbation difference spectroscopy, chemical modification, and mutagenesis

AU - Svensson, Birte

AU - Sierks, Michael

PY - 1992/4/6

Y1 - 1992/4/6

N2 - The roles of the aromatic side chains of the glucoamylase from Aspergillus niger in the binding of ligands, as determined by differences spectroscopy using four types of inhibitors (a) valienamine-derived, (b) 1-deoxynojrimyscins, (c) d-glucono-1,5-lactone, and (d), two types of disaccharide substrates (a) α-(1»4)-linked and (b) α-(1»6)-linked, and three cyclomalto-oligosaccharides (cyclodextrins, CDs) are discussed. An unusual change in absorbance from 300 to 310-320 nm, obtained only with valienamine-derived inhibitors or when d-glucono-1,5-lactone and maltose are combined, is concluded to arise when subsite 2 is occupied in a transition-state-type of complex. The single mutations of two residues thought to be involved in binding, namely, Tyr116»Ala and Trp120»Phe, alter, but do not abolish this perturbation. The perturbations in the spectra also suggest that maltose and isomaltose have different modes of binding. The following Kd values (m) were determined: acarbose, < x 10-12; methyl acarviosinide, 1.6 x 10-6; and the d-gluco and l-ido forms of hydrogenated acarbose, 1.4 x 10-8 and 5.2 x 10-6, respectively. Therefore, both the valienamine moiety and the chain length of acarbose are important for tight binding. In contrast to the valienamine-derived inhibitors, none of the 1-deoxynojirimycin type protected glucoamylase against inactivating oxidation of tryptophyanyl residues, although each had a Kd value of ∼ 4 x 10-6m. There are two distinct carbohydrate-binding areas in glucoamylase, namely, the active site in the catalytic domain and a strach-granule-binding site in the C-terminal domain. The α-,β-, and γ-CDs have high affinity for the starch-binding domain and low affinity for the active site, whereas the reverse was found for acarbose.

AB - The roles of the aromatic side chains of the glucoamylase from Aspergillus niger in the binding of ligands, as determined by differences spectroscopy using four types of inhibitors (a) valienamine-derived, (b) 1-deoxynojrimyscins, (c) d-glucono-1,5-lactone, and (d), two types of disaccharide substrates (a) α-(1»4)-linked and (b) α-(1»6)-linked, and three cyclomalto-oligosaccharides (cyclodextrins, CDs) are discussed. An unusual change in absorbance from 300 to 310-320 nm, obtained only with valienamine-derived inhibitors or when d-glucono-1,5-lactone and maltose are combined, is concluded to arise when subsite 2 is occupied in a transition-state-type of complex. The single mutations of two residues thought to be involved in binding, namely, Tyr116»Ala and Trp120»Phe, alter, but do not abolish this perturbation. The perturbations in the spectra also suggest that maltose and isomaltose have different modes of binding. The following Kd values (m) were determined: acarbose, < x 10-12; methyl acarviosinide, 1.6 x 10-6; and the d-gluco and l-ido forms of hydrogenated acarbose, 1.4 x 10-8 and 5.2 x 10-6, respectively. Therefore, both the valienamine moiety and the chain length of acarbose are important for tight binding. In contrast to the valienamine-derived inhibitors, none of the 1-deoxynojirimycin type protected glucoamylase against inactivating oxidation of tryptophyanyl residues, although each had a Kd value of ∼ 4 x 10-6m. There are two distinct carbohydrate-binding areas in glucoamylase, namely, the active site in the catalytic domain and a strach-granule-binding site in the C-terminal domain. The α-,β-, and γ-CDs have high affinity for the starch-binding domain and low affinity for the active site, whereas the reverse was found for acarbose.

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