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, M. R. Sierks

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Abstract

The roles of the aromatic side chains of the glucoamylase from Aspergillus niger in the binding of ligands, as determined by difference spectroscopy using four types of inhibitors (a) valienamine-derived, (b) 1-deoxynojirimycins, (c) D-glucono-1,5-lactone, and (d) maltitol, two types of disaccharide substrates (a) alpha-(1----4)-linked and (b) alpha-(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 the 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, less than 6 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 tryptophanyl residues, although each had a Kd value of approximately 4 x 10(-6) M. There are two distinct carbohydrate-binding areas in glucoamylase, namely, the active site in the catalytic domain and a starch-granule-binding site in the C-terminal domain. The alpha-, beta-, and gamma-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
JournalCarbohydrate Research
Volume228
Pages (from-to)29-44
ISSN0008-6215
DOIs
Publication statusPublished - 1992
Externally publishedYes

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