Formation of covalent β-linked carbohydrate–enzyme intermediates during the reactions catalyzed by α-amylases Original Research Article

Porcine pancreatic and Bacillus amyloliquefaciens α-amylases were examined for the formation of covalent carbohydrate intermediates during reaction. The enzymes were precipitated and denatured by adding 10 volumes of acetone. When these denatured enzymes were mixed with methyl α-6-[3H]-maltooligosaccharide glycosides and chromatographed on BioGel P-2, no carbohydrate was found in the protein void volume peak. When the enzymes were added to the methyl α-6-[3H]-maltooligosaccharide glycosides and allowed to react for 15 s at 1 °C and then precipitated and denatured with 10 volumes of acetone, 3H-labeled carbohydrates were found in the BioGel P-2 protein void volume peak, indicating the formation of enzyme–carbohydrate covalent intermediates. 1H NMR analysis of the denatured enzyme from the reaction with methyl α-maltooligosaccharide glycosides confirmed that carbohydrate was attached to the denatured enzyme. 1H NMR saturation-transfer analysis further showed that the carbohydrate was attached to the denatured enzyme by a β-configuration. This configuration is what would be expected for an enzyme that catalyzes the hydrolysis of α-(1→4) glycosidic linkages by a two-step, SN2 double-displacement reaction to give retention of the α-configuration of the substrates at the reducing-end of the products.