Difference spectroscopic study of the interaction between soybean beta-amylase and substrate or substrate analogues.
1. In order to investigate the interactions between soybean beta-amylase [EC 3.2.1.2] and ligands (maltotriose as substrate, and maltose and alpha- and beta-cyclodextrins as inhibitors for the hydrolysis of maltoheptaose), the difference spectra were measured at 25 degrees C and pH 5.4, in 0.05 M acetate buffer. Each difference spectrum produced by these ligands showed a clear peak at 292-293 nm due to a tryptophan residue. In addition to this peak, the spectra of alpha- and beta-cyclodextrins showed a specific peak at 298-299 nm, and that of maltotriose showed a shoulder at 298 nm. 2. From the concentration dependency of the difference molar extinction delta epsilon, at 292-293 nm or at 298-299 nm, the dissociation constant of the enzyme-ligand complex, Kd, was evaluated for maltotriose, and alpha- and beta-cyclodextrins. For each ligand, the Kd values obtained at these two wavelengths were in good agreement with Michaelis constant, Km, or the inhibitor constant, Ki. The Kd value for maltose obtained from the titration of delta epsilon at 292 nm was also in good agreement with Ki. 3. Maltose produced a hydrophobic change in the environment of the tryptophan residue, while the interactions of maltotriose, and alpha- and beta-cyclodextrins with this enzyme caused an electrostatic change in the vicinity of the tryptophan residue in addition to the hydrophobic change. Since the signal at 298-299 nm was not found in the difference spectrum of maltose, this signal may be due to a tryptophan residue different from that which produces the signal at 292-293 nm. If both the signals are due to the same tryptophan residue, we must conclude that some conformational change is caused in the enzyme active site by the ligand binding.
D000681:Amylases / Q000378:metabolism*; D003505:Cyclodextrins / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D013025:Glycine max; D013053:Spectrophotometry / Q000379:methods; D013379:Substrate Specificity; D013997:Time Factors; D014312:Trisaccharides / Q000378:metabolism; D047391:alpha-Cyclodextrins; D001614:beta-Amylase / Q000378:metabolism*; D047392:beta-Cyclodextrins
D003505:Cyclodextrins; D008024:Ligands; D014312:Trisaccharides; D047391:alpha-Cyclodextrins; D047392:beta-Cyclodextrins; C008317:maltotriose; D000681:Amylases; D001614:beta-Amylase; C031215:betadex; C032613:alpha-cyclodextrin