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±0.85kJ/mol) and between 1,6-linkage and 1,3-linkage (ΔΔG‡0=1.46±0.23kJ/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±0.47kJ/mol higher in free energy than the 1,3-linkage, and the 1,6-linkage 4.44±0.71kJ/mol lower.