The method of integrated kinetics and its applicability to the exo-glycosidase-catalyzed hydrolysis of p-nitrophenyl glycosides

• Simple analysis of integrated kinetics provides kcat, Km, KI in a single experiment.
• Mutarotation of the liberated carbohydrate product affects the reaction course.
• Temperature dependence of galactose mutarotaion is characterized.
• Inhibition by α- and β-galactoses is distinguishable by temperature change.

In the present work we suggest an efficient method, using the whole time course of the reaction, whereby parameters kcat, Km and product KI for the hydrolysis of a p-nitrophenyl glycoside by an exo-acting glycoside hydrolase can be estimated in a single experiment. Its applicability was demonstrated for three retaining exo-glycoside hydrolases, β-xylosidase from Aspergillus awamori, β-galactosidase from Penicillium sp. and α-galactosidase from Thermotoga maritima (TmGalA). During the analysis of the reaction course catalyzed by the TmGalA enzyme we had observed that a non-enzymatic process, mutarotation of the liberated α-d-galactose, affected the reaction significantly.

Figure optionsDownload as PowerPoint slide