title: The method of integrated kinetics and its applicability to the exo-glycosidase-catalyzed hydrolysis of p-nitrophenyl glycosides.
abstract: 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.
authors.affiliation: National Research Center "Kurchatov Institute", B.P. Konstantinov Petersburg Nuclear Physics Institute, 188300, Gatchina, Orlova roscha, Russia; Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences, Uppsala, Sweden.
authors.forename: Anna S
authors.initials: AS
authors.lastname: Borisova
authors.affiliation: Department of Biochemistry and Structural Biology, Lund University, Lund S-221 00, Sweden.
authors.forename: Sumitha K
authors.initials: SK
authors.lastname: Reddy
authors.affiliation: National Research Center "Kurchatov Institute", B.P. Konstantinov Petersburg Nuclear Physics Institute, 188300, Gatchina, Orlova roscha, Russia.
authors.forename: Dina R
authors.initials: DR
authors.lastname: Ivanen
authors.affiliation: National Research Center "Kurchatov Institute", B.P. Konstantinov Petersburg Nuclear Physics Institute, 188300, Gatchina, Orlova roscha, Russia.
authors.forename: Kirill S
authors.initials: KS
authors.lastname: Bobrov
authors.affiliation: National Research Center "Kurchatov Institute", B.P. Konstantinov Petersburg Nuclear Physics Institute, 188300, Gatchina, Orlova roscha, Russia.
authors.forename: Elena V
authors.initials: EV
authors.lastname: Eneyskaya
authors.affiliation: National Research Center "Kurchatov Institute", B.P. Konstantinov Petersburg Nuclear Physics Institute, 188300, Gatchina, Orlova roscha, Russia; St. Petersburg State Polytechnical University, 29 Politekhnicheskaya st., 195251 St. Petersburg, Russia.
authors.forename: Georgy N
authors.initials: GN
authors.lastname: Rychkov
authors.affiliation: Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences, Uppsala, Sweden.
authors.forename: Mats
authors.initials: M
authors.lastname: Sandgren
authors.affiliation: Department of Biochemistry and Structural Biology, Lund University, Lund S-221 00, Sweden.
authors.forename: Henrik
authors.initials: H
authors.lastname: Stålbrand
authors.affiliation: Department of Chemical Sciences, University of Huddersﬁeld, Queensgate, Huddersﬁeld HD1 3DH, UK.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Sinnott
authors.affiliation: National Research Center "Kurchatov Institute", B.P. Konstantinov Petersburg Nuclear Physics Institute, 188300, Gatchina, Orlova roscha, Russia; St. Petersburg State Polytechnical University, 29 Politekhnicheskaya st., 195251 St. Petersburg, Russia. Electronic address: kulm@omrb.pnpi.spb.ru.
authors.forename: Anna A
authors.initials: AA
authors.lastname: Kulminskaya
authors.affiliation: National Research Center "Kurchatov Institute", B.P. Konstantinov Petersburg Nuclear Physics Institute, 188300, Gatchina, Orlova roscha, Russia; St. Petersburg State Polytechnical University, 29 Politekhnicheskaya st., 195251 St. Petersburg, Russia.
authors.forename: Konstantin A
authors.initials: KA
authors.lastname: Shabalin
mesh_terms: D001230:Aspergillus / Q000737:chemistry*; D005690:Galactose / Q000737:chemistry; D006027:Glycosides / Q000737:chemistry*; D006868:Hydrolysis; D007700:Kinetics; D010407:Penicillium / Q000737:chemistry*; D020124:Thermotoga maritima / Q000737:chemistry*; D014995:Xylosidases / Q000737:chemistry; D000519:alpha-Galactosidase / Q000737:chemistry*; D001616:beta-Galactosidase / Q000737:chemistry*
chemical_list: D006027:Glycosides; D014995:Xylosidases; D000519:alpha-Galactosidase; D001616:beta-Galactosidase; C026579:exo-1,4-beta-D-xylosidase; D005690:Galactose
keywords: Integrated kinetics; Mutarotation; Retaining glycoside hydrolase