title: Active-site- and substrate-specificity of Thermoanaerobium Tok6-B1 pullulanase.
abstract: Thermoanaerobium Tok6-B1 pullulanase (EC 3.2.1.41) was active on alpha 1-6-glucosidic linkages of pullulan, amylopectin and glycogen and the alpha 1-4 linkages of amylose, amylopectin and glycogen but not of pullulan. Hydrolysis of short-chain-length malto-oligosaccharides (seven or fewer glucose residues) yielded maltose as product. Pullulan hydrolysis was pH-dependent and a plot of log(V/Km) versus pH implied a carboxy group with pKa 4.3 at the active site. Modification with 1-(3-dimethylaminopropyl)-3-ethylcarbodi-imide (EDAC) confirmed this view, and analysis of the order of reaction and inactivation kinetics suggested the presence of a single carboxy group at a catalytic centre of the active site. EDAC-mediated inhibition of pullulan alpha 1-6-bond hydrolysis was relieved by amylose or pullulan. Similarly both pullulan and amylose protected the activity directed at alpha 1-4 bonds of amylose from EDAC inhibition. When both amylose and pullulan were simultaneously present, the observed rate of product formation closely fitted a kinetic model in which both substrates were hydrolysed at the same active site.
mesh_terms: D000688:Amylose / Q000378:metabolism; D001421:Bacteria, Anaerobic / Q000201:enzymology*; D001665:Binding Sites; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D009844:Oligosaccharides / Q000378:metabolism; D013379:Substrate Specificity; D000516:alpha-Amylases / Q000378:metabolism
chemical_list: D009844:Oligosaccharides; D000688:Amylose; D006026:Glycoside Hydrolases; D000516:alpha-Amylases; C100181:pullulanase; D005022:Ethyldimethylaminopropyl Carbodiimide