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.