Formation of covalent beta-linked carbohydrate-enzyme intermediates during the reactions catalyzed by alpha-amylases

Porcine pancreatic and Bacillus amyloliquefaciens alpha-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 alpha-6-[(3)H]-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 alpha-6-[(3)H]-maltooligosaccharide glycosides and allowed to react for 15s at 1 degrees C and then precipitated and denatured with 10 volumes of acetone, (3)H-labeled carbohydrates were found in the BioGel P-2 protein void volume peak, indicating the formation of enzyme-carbohydrate covalent intermediates. (1)H NMR analysis of the denatured enzyme from the reaction with methyl alpha-maltooligosaccharide glycosides confirmed that carbohydrate was attached to the denatured enzyme. (1)H NMR saturation-transfer analysis further showed that the carbohydrate was attached to the denatured enzyme by a beta-configuration. This configuration is what would be expected for an enzyme that catalyzes the hydrolysis of alpha-(1-->4) glycosidic linkages by a two-step, S(N)2 double-displacement reaction to give retention of the alpha-configuration of the substrates at the reducing-end of the products.