The method of integrated kinetics and its applicability to the exo-glycosidase-catalyzed hydrolysis of p-nitrophenyl glycosides. (14th August 2015)

• Record Type: Journal Article
• Authors: Borisova, Anna S.; Reddy, Sumitha K.; Ivanen, Dina R.; Bobrov, Kirill S.; Eneyskaya, Elena V.; Rychkov, Georgy N.; Sandgren, Mats; Stålbrand, Henrik; Sinnott, Michael L.; Kulminskaya, Anna A.; Shabalin, Konstantin A.
• Is Part Of: Carbohydrate research. Volume 412(2015)
• Page Start: 43
• Page End: 49
• Publication Date: 2015-08-14
• Subjects: Integrated kinetics -- Retaining glycoside hydrolase -- Mutarotation; TmGalA α-galactosidase from Thermotoga maritima -- pNP para-nitrophenyl -- pNPαGal pNP-α-d-galactopyranoside -- pNPXyl p-nitrophenyl β-xylopyranoside -- pNPβGal p-nitrophenyl β-galacopyranoside; Carbohydrates -- Periodicals; Chemistry, Organic -- Periodicals; Biochemistry -- Periodicals; Carbohydrates -- Periodicals; Chimie organique -- Périodiques; Glucides -- Périodiques; Biochemistry; Carbohydrates; Chemistry, Organic; Periodicals; Electronic journals; 507.78
• DOI: 10.1016/j.carres.2015.03.021
• Languages: English
• ISSNs: 0008-6215

Abstract

Abstract: In the present work we suggest an efficient method, using the whole time course of the reaction, whereby parameters k cat, K m and product K I 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. Graphical abstract: Highlights: Simple analysis of integrated kinetics provides k cat, K m, K I 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.