title: α-Galactobiosyl units: thermodynamics and kinetics of their formation by transglycosylations catalysed by the GH36 α-galactosidase from Thermotoga maritima.
abstract: Broad regioselectivity of α-galactosidase from Thermotoga maritima (TmGal36A) is a limiting factor for application of the enzyme in the directed synthesis of oligogalactosides. However, this property can be used as a convenient tool in studies of thermodynamics of a glycosidic bond. Here, a novel approach to energy difference estimation is suggested. Both transglycosylation and hydrolysis of three types of galactosidic linkages were investigated using total kinetics of formation and hydrolysis of pNP-galactobiosides catalysed by monomeric glycoside hydrolase family 36 α-galactosidase from T. maritima, a retaining exo-acting glycoside hydrolase. We have estimated transition state free energy differences between the 1,2- and 1,3-linkage (ΔΔG(‡)0 values were equal 5.34 ± 0.85 kJ/mol) and between 1,6-linkage and 1,3-linkage (ΔΔG(‡)0=1.46 ± 0.23 kJ/mol) in pNP-galactobiosides over the course of the reaction catalysed by TmGal36A. Using the free energy difference for formation and hydrolysis of glycosidic linkages (ΔΔG(‡)F-ΔΔG(‡)H), we found that the 1,2-linkage was 2.93 ± 0.47 kJ/mol higher in free energy than the 1,3-linkage, and the 1,6-linkage 4.44 ± 0.71 kJ/mol lower.
authors.affiliation: National Research Center "Kurchatov Institute", B.P. Konstantinov Petersburg Nuclear Physics Institute, Orlova Roscha, 188300 Gatchina, 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: National Research Center "Kurchatov Institute", B.P. Konstantinov Petersburg Nuclear Physics Institute, Orlova Roscha, 188300 Gatchina, 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, Orlova Roscha, 188300 Gatchina, 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, Orlova Roscha, 188300 Gatchina, 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, Orlova Roscha, 188300 Gatchina, Russia; St. Petersburg State Polytechnical University, 29 Politechnicheskaya str., 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: National Research Center "Kurchatov Institute", B.P. Konstantinov Petersburg Nuclear Physics Institute, Orlova Roscha, 188300 Gatchina, Russia; St. Petersburg State Polytechnical University, 29 Politechnicheskaya str., 195251 St. Petersburg, Russia. Electronic address: kulm@omrb.pnpi.spb.ru.
authors.forename: Anna A
authors.initials: AA
authors.lastname: Kulminskaya
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, Orlova Roscha, 188300 Gatchina, Russia; St. Petersburg State Polytechnical University, 29 Politechnicheskaya str., 195251 St. Petersburg, Russia.
authors.forename: Konstantin A
authors.initials: KA
authors.lastname: Shabalin
mesh_terms: D055162:Biocatalysis; D004187:Disaccharides / Q000737:chemistry* / Q000378:metabolism*; D006031:Glycosylation; D006868:Hydrolysis; D007700:Kinetics; D013237:Stereoisomerism; D013379:Substrate Specificity; D020124:Thermotoga maritima / Q000201:enzymology*; D000519:alpha-Galactosidase / Q000378:metabolism*
chemical_list: D004187:Disaccharides; C056853:6-O-galactopyranosylgalactose; D000519:alpha-Galactosidase
keywords: Free energy differences; Kinetics; Regioselectivity; Thermotoga maritima; Transglycosylation; α-Galactosidase