蜡样芽孢杆菌蕈状变种β-淀粉酶活性位点结构的动力学研究

Kinetic Study of the Active Site Structure of β-Amylase from Bacillus cereus var. mycoides.

作者信息

Nitta Y, Shirakawa M, Takasaki Y

机构信息

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.

出版信息

Biosci Biotechnol Biochem. 1996 Jan;60(5):823-7. doi: 10.1271/bbb.60.823.

DOI:10.1271/bbb.60.823

PMID:27281142

Abstract

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.

摘要

基于Hiromi理论，以¹⁴C放射性标记的麦芽寡糖为底物，对蜡状芽孢杆菌蕈状变种β -淀粉酶活性位点的亚位点亲和力进行了评估。据估计，该活性位点由六个亚位点组成，所有亚位点的亲和力均可评估。该活性位点与大豆和麦麸β -淀粉酶的活性位点具有相同的亚位点排列方式。α-1,4糖苷键的固有分解速率常数（kint）是其他酶的五到七倍。根据log[k0/Km]对pH的依赖性，两个功能性可电离基团的pK值分别为pK1 = 4.0和pK2 = 8.4。这两个pK值比其他酶的pK1大0.5 - 0.6个单位，比pK2大0.2 - 0.3个单位。对于用2,3 - 环氧丙基α - D -吡喃葡萄糖苷（α - EPG）对该酶进行亲和标记，α - EPG的结合亲和力比其他β -淀粉酶高1 - 1.6千卡/摩尔。