α-Galactobiosyl units: thermodynamics and kinetics of their formation by transglycosylations catalysed by the GH36 α-galactosidase from Thermotoga maritima

Broad regioselectivity of a-galactosidase from Thermotoga maritima (pTmGal36A) 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-galacto-biosides catalysed by monomeric glycoside hydrolase family 36 a-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 (Delta Delta G(0)(double dagger) values were equal 5.34 +/- 0.85 kJ/ mol) and between 1,6-linkage and 1,3-linkage (Delta Delta G(0)(double dagger) = 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 (Delta Delta G(F)(double dagger) -Delta Delta G(H)(double dagger)), 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. (C) 2014 Elsevier Ltd. All rights reserved.