Kinetic Study of the Active Site Structure of β-Amylase from Bacillus cereus var. mycoides.
The subsite affinities of the active site of β-amylase from Bacillus cereus var. mycoides were evaluated based on Hiromi's theory, using (14)C-radiolabeled maltooligosaccharides as substrate. It was estimated that the active site consisted of six subsites, and all subsite affinities could be evaluated. The active site had a common subsite arrangement with those of β -amylases from soybean and wheat bran. The intrinsic breakdown rate constant of α-1,4 glucosidic linkage (kint) was five to seven times as large as those of the other enzymes.From the pH dependence of log[k0/Km], pK values of two functional ionizable groups were pK1 =4.0 and pK2 = 8.4. The pK values were 0.5-0.6 units for pK1 and 0.2-0.3 units for pK2 larger than those of the other enzymes. For the affinity-labeling of this enzyme by 2, 3 epoxypropyl α-D-glucopyranoside (α-EPG), the binding affinity of α-EPG was 1-1.6kcal/mol larger than those of the other β-amylases.
a Laboratory of Biophysical Chemistry, College of Agriculture, Osaka Prefecture University , 1-1 Gakuen-cho, Sakai, Osaka 593 , Japan.
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Nitta
a Laboratory of Biophysical Chemistry, College of Agriculture, Osaka Prefecture University , 1-1 Gakuen-cho, Sakai, Osaka 593 , Japan.|b Department of Materials Science , Faculty of Engineering, Miyazaki University , 1-1 Nishi, Gakuen-kibanadai, Miyazaki 889-21 , Japan.
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Shirakawa
b Department of Materials Science , Faculty of Engineering, Miyazaki University , 1-1 Nishi, Gakuen-kibanadai, Miyazaki 889-21 , Japan.
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Takasaki
Bacillus cereus var. mycoides; active site; functional group; subsite affinity; β-amylase