Interaction of native and SH-modified beta-amylase of soybean with cyclohexadextrin and maltose.
Cyclohexadextrin and maltose bound to soybean beta-amylase and affected the environments of tryptophan and tyrosine residues, producing characteristic difference spectra in the ultraviolet region. The difference spectrum produced by cyclohexadextrin, a competitive inhibitor, had peaks at 285, 292, and 299 nm, while that by maltose, a reaction product, had peaks at 285 and 292 nm and a small trough at around 300 nm. By using the peaks at 292 and 299 nm, the dissociation constants of enzyme-cyclohexadextrin and enzyme-maltose complexes were calculated to be 0.35 mM and 8.1 mM, respectively. The effects of modification of SH groups of beta-amylase on the interaction of the enzyme with these sugars were examined by using beta-amylase carboxymethylated at the SH1 site and the enzyme modified at SH1 and SH2 sites with iodoacetamide or with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). The dissociation constants of the enzyme-cyclohexadextrin and enzyme-maltose complexes were not changed by the modification of these SH groups, but the modification of SH2, the so-called essential SH group of soybean beta-amylase, strongly affected the difference spectra produced by maltose. The spectrophotometric titration of beta-amylase by cyclohexadextrin in the presence of maltose showed that cyclohexadextrin and maltose bind to the enzyme competitively, regardless of the modification of SH2. These results indicated that SH2 is located near the binding site of cyclohexadextrin and maltose, but is not involved in the binding of these sugars.
D000681:Amylases / Q000032:analysis*; D001667:Binding, Competitive; D002942:Circular Dichroism; D003505:Cyclodextrins / Q000032:analysis*; D003912:Dextrins / Q000032:analysis*; D007700:Kinetics; D008320:Maltose / Q000032:analysis*; D011485:Protein Binding; D013025:Glycine max / Q000201:enzymology; D013056:Spectrophotometry, Ultraviolet; D013213:Starch / Q000032:analysis*; D013438:Sulfhydryl Compounds; D001614:beta-Amylase / Q000032:analysis* / Q000037:antagonists & inhibitors
D003505:Cyclodextrins; D003912:Dextrins; D013438:Sulfhydryl Compounds; D008320:Maltose; D013213:Starch; D000681:Amylases; D001614:beta-Amylase