title: Interaction of wheat β-amylase with maltose and glucose as examined by fluorescence.
abstract: Fluorescence of wheat β-amylase (WBA) was quenched by the interaction with maltose or glucose, which are competitive inhibitors of WBA, suggesting that the states of tryptophan and tyrosine residues could be changed by the interaction. The fluorescence emitted by excitation at 280 and 295 nm was titrated by changing the concentrations of maltose and glucose. The dissociation constant (Kd) values of the WBA-maltose and WBA-glucose complexes were determined to be 0.20 ± 0.12 M for maltose and 0.36 ± 0.11 M for glucose at 25°C, pH 5.4. Maltose exhibited additional binding mode at higher concentration with a distinct Kd value (1.5 ± 0.4 M). The Kd values at various temperatures and pHs are in agreement with the inhibitor constant (Ki) values previously reported. The negative standard enthalpy changes (ΔH°) of the WBA association with glucose and maltose indicate that the associations are exothermic. The association constant (Ka) and ΔG° values of the maltose and glucose binding to WBA decreased slightly with increasing temperature from 25°C to 45°C but not dependent on pH change (pH 3.0, 5.4 and 9.0). Fluorescence of WBA could be used as a structural probe to examine the inhibitory interaction with the products of starch hydrolysis.
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Tadessa
authors.initials: T
authors.lastname: Daba
authors.forename: Kenji
authors.initials: K
authors.lastname: Kojima
authors.forename: Kuniyo
authors.initials: K
authors.lastname: Inouye
mesh_terms: D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013213:Starch / Q000378:metabolism; D013816:Thermodynamics; D014908:Triticum / Q000201:enzymology*; D014364:Tryptophan / Q000737:chemistry; D001614:beta-Amylase / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D010940:Plant Proteins; D008320:Maltose; D014364:Tryptophan; D013213:Starch; D001614:beta-Amylase; D005947:Glucose
keywords: dissociation constant; fluorescence quenching; glucose; maltose; wheat β-amylase