Nishimoto M, Kubota M, Tsuji M, Mori H, Kimura A, Matsui H, Chiba S
Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo, Japan.
Biosci Biotechnol Biochem. 2001 Jul;65(7):1610-6. doi: 10.1271/bbb.65.1610.
Alpha-glucosidase III, which was different in substrate specificity from honeybee alpha-glucosidases I and II, was purified as an electrophoretically homogeneous protein from honeybees, by salting-out chromatography, DEAE-cellulose, DEAE-Sepharose CL-6B, Bio-Gel P-150, and CM-Toyopearl 650M column chromatographies. The enzyme preparation was confirmed to be a monomeric protein and a glycoprotein containing about 7.4% of carbohydrate. The molecular weight was estimated to approximately 68,000, and the optimum pH was 5.5. The substrate specificity of alpha-glucosidase III was kinetically investigated. The enzyme did not show unusual kinetics, such as the allosteric behaviors observed in alpha-glucosidases I and II, which are monomeric proteins. The enzyme was characterized by the ability to rapidly hydrolyze sucrose, phenyl alpha-glucoside, maltose, and maltotriose, and by extremely high Km for substrates, compared with those of alpha-glucosidases I and II. Especially, maltotriose was hydrolyzed over 3 times as rapidly as maltose. However, maltooligosaccharides of four or more in the degree of polymerization were slowly degraded. The relative rates of the k0 values for maltose, sucrose, p-nitrophenyl alpha-glucoside and maltotriose were estimated to be 100, 527, 281 and 364, and the Km values for these substrates, 11, 30, 13, and 10 mM, respectively. The subsite affinities (Ai's) in the active site were tentatively evaluated from the rate parameters for maltooligosaccharides. In this enzyme, it was peculiar that the Ai value at subsite 3 was larger than that of subsite 1.
α-葡萄糖苷酶III在底物特异性上与蜜蜂α-葡萄糖苷酶I和II不同，通过盐析色谱法、DEAE-纤维素柱色谱法、DEAE-琼脂糖CL-6B柱色谱法、Bio-Gel P-150柱色谱法和CM-Toyopearl 650M柱色谱法从蜜蜂中纯化得到一种电泳纯的蛋白质。该酶制剂被确认为单体蛋白和一种含糖量约为7.4%的糖蛋白。估计其分子量约为68,000，最适pH为5.5。对α-葡萄糖苷酶III的底物特异性进行了动力学研究。该酶没有表现出异常的动力学，如在单体蛋白α-葡萄糖苷酶I和II中观察到的别构行为。该酶的特点是能够快速水解蔗糖、苯基α-葡萄糖苷、麦芽糖和麦芽三糖，并且与α-葡萄糖苷酶I和II相比，对底物的Km值极高。特别是，麦芽三糖的水解速度比麦芽糖快3倍以上。然而，聚合度为四个或更多的麦芽寡糖降解缓慢。麦芽糖、蔗糖、对硝基苯基α-葡萄糖苷和麦芽三糖的k0值相对速率估计分别为100、527、281和364，这些底物的Km值分别为11、30、13和10 mM。根据麦芽寡糖的速率参数初步评估了活性位点中的亚位点亲和力（Ai值）。在这种酶中，亚位点3的Ai值大于亚位点1的Ai值是其独特之处。
