3rd meeting of young researchers at UP 1 - IJUP - Universidade do ...

Computational Studies on the β-Galactosidase Catalytic Mechanism:
Hydrolysis and Transglycosylation Reactions
N.F. Brás, P.A. Fernandes and M.J. Ramos
Department of Chemistry, Faculty of Sciences, University of Porto, Portugal.
Galactooligosaccharides are functional food ingredients, combining prebiotic properties
beneficial to the human health with valuable physico-chemical properties in food processing [1] .
Its production is carried out by β-galactosidase, which simultaneously catalyses the hydrolysis
reaction of lactose into simple sugars [2] . This enzymatic catalyst is extremely important for the
synthesis and modification of carbohydrates, which have great interest to the food and
pharmaceutical industries given their potential application in therapeutics.
In this work we have used computational methods to study the catalytic mechanism of
hydrolysis and a set of distinct transglycosylation reactions of a retaining galactosidase, with
atomic detail, with lactose as the natural substrate. The ONIOM method
(BB1K:AMBER//B3LYP:AMBER calculations) was employed to deal with such a large
enzymatic system. Such methodology can efficiently account for the stereochemistry of the
reactive residues, as well as the long range enzyme-substrate interactions. The possible
importance of the magnesium ion in the catalytic reaction was investigated and was found that
indeed it catalyses the transformation, lowering the activation barrier by 14.9 kcal/mol. The
calculations have shown that the formation of ß(1-3) glycosidic linkages is thermodynamically
very unfavorable. In contrast, the formation of ß(1-6) glycosidic bonds is the most favored, in
total agreement with the enantioselectivity experimentally observed.
The data have also clearly shown the importance of the enzyme scaffold beyond the first shell
aminoacids in the stabilization of the transition states. It is fundamental to include explicitly the
enzyme in computational studies.
References:
[1] Brás, N.F., Fernandes, P.A. and Ramos, M.J. (2009), Theoretical Chemistry Accounts, 2009,
122:283–296.
[2] Brás, N.F., Moura-Tamames, S.A., Fernandes, P.A. and Ramos M.J. (2008), Journal of
Computational Chemistry, Vol. 29, No. 15.
[3] Brás, N.F., Fernandes, P.A. and Ramos, M.J. (2010), Journal of Chemical Theory and Computation.
162 3 rd meeting of young researchers at UP