Active-site- and substrate-specificity of Thermoanaerobium Tok6-B1 pullulanase

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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 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.