Thermoanaerobium Tok6-B1 pullulanase (EC 126.96.36.199) 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.
Research Article| September 01 1987
Active-site- and substrate-specificity of Thermoanaerobium Tok6-B1 pullulanase
A R Plant;
R M Clemens;
H W Morgan;
Biochem J (1987) 246 (2): 537–541.
- Views Icon Views
- Share Icon Share
A R Plant, R M Clemens, H W Morgan, R M Daniel; Active-site- and substrate-specificity of Thermoanaerobium Tok6-B1 pullulanase. Biochem J 1 September 1987; 246 (2): 537–541. doi: https://doi.org/10.1042/bj2460537
Download citation file: