title: Detection of a covalent intermediate in the mechanism of action of porcine pancreatic alpha-amylase by using 13C nuclear magnetic resonance.
abstract: The catalytic mechanism of porcine pancreatic alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) has been examined by nuclear magnetic resonance (NMR) at subzero temperatures by using [1-13C]maltotetraose as substrate. Spectral summation and difference techniques revealed a broad resonance peak, whose chemical shift, relative signal intensity and time-course appearance corresponded to a beta-carboxyl-acetal ester covalent enzyme-glycosyl intermediate. This evidence supports a double-displacement covalent mechanism for porcine pancreatic alpha-amylase-catalyzed hydrolysis of glycosidic linkages, based on the presence of catalytic aspartic acid residues within the active site of this enzyme.
authors: Department of Chemical Engineering, Iowa State University, Ames 50011.
authors: B Y
authors: BY
authors: Tao
authors: P J
authors: PJ
authors: Reilly
authors: J F
authors: JF
authors: Robyt
mesh_terms: D000818:Animals; D002247:Carbon Isotopes; D004121:Dimethyl Sulfoxide; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D008320:Maltose / Q000031:analogs & derivatives / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism; D010179:Pancreas / Q000201:enzymology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013552:Swine; D013696:Temperature; D000516:alpha-Amylases
chemical_list: D002247:Carbon Isotopes; D009844:Oligosaccharides; D008320:Maltose; C009819:maltotetraose; D000516:alpha-Amylases; D004121:Dimethyl Sulfoxide