Interaction of soybean beta-amylase with glucose.
The interaction of soybean beta-amylase with glucose was investigated by inhibition kinetics studies and spectroscopic measurements. The inhibition type, inhibitor constant (Ki) and dissociation constant (Kd) of beta-amylase-glucose complex were dependent on pH. At pH 8.0, glucose behaved as a competitive inhibitor (Ki = 34 mM). Binding of glucose produced a characteristic difference spectrum and a change of circular dichroism (CD) at pH 8.1. By using difference absorbance at 292 nm and difference ellipticity at 290 nm, Kd values for beta-amylase-glucose complex were determined to be 45 and 46 mM, respectively. In contrast to pH 8.0, glucose behaved as a mixed-type inhibitor (Ki = 320 mM) at pH 5.4. The Kd values obtained from the difference spectrum were increased by lowering the pH from 8. The pH dependence of the Ki and Kd values suggested that one ionizable group of pK = 8.0, which is shifted to 6.9 by the binding of glucose, controls the binding affinity of glucose. The binding of glucose competed with the binding of cyclohexaamylose and maltose at pH 8.0. The modification of SH groups of the enzyme affected the binding of glucose but did not affect the binding of maltose or cyclohexaamylose at pH 8.0. It was concluded from these results that the binding site of glucose is different from that of maltose and cyclohexaamylose. Presumably, glucose may bind to the subsite 1 of soybean beta-amylase.
D000681:Amylases / Q000378:metabolism*; D001667:Binding, Competitive; D055598:Chemical Phenomena; D002621:Chemistry; D002942:Circular Dichroism; D003505:Cyclodextrins / Q000378:metabolism / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism* / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008320:Maltose / Q000378:metabolism / Q000494:pharmacology; D013025:Glycine max / Q000201:enzymology*; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds; D047391:alpha-Cyclodextrins; D001614:beta-Amylase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D003505:Cyclodextrins; D013438:Sulfhydryl Compounds; D047391:alpha-Cyclodextrins; D008320:Maltose; D000681:Amylases; D001614:beta-Amylase; D005947:Glucose; C032613:alpha-cyclodextrin