QM/MM Studies on the β-Galactosidase Catalytic Mechanism: Hydrolysis and Transglycosylation Reactions.

Carbohydrates perform a wide range of crucial functions in biological systems and are of great interest for the food and pharmaceutical industries. β-Galactosidase from Escherichia coli catalyzes both the hydrolytic breaking of the very stable glycosidic bond of lactose and a series of transglycosylation reactions. These reactions are crucial for the development of new carbohydrate molecules, as well as the optimization of their syntheses. 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 address such a large enzymatic system. Such a 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 it was found that, indeed, the magnesium ion catalyzes the transformation, lowering the activation barrier by 14.9 kcal/mol. The calculations indicate that the formation of β(1-3) glycosidic linkages is thermodynamically very unfavorable. In contrast, the formation of β(1-6) glycosidic bonds is the most favored, in complete agreement with the enantioselectivity observed experimentally. The data also clearly show the importance of the enzyme scaffold beyond the first-shell amino acids in the stabilization of the transition states. It is fundamental to include the enzyme explicitly in computational studies.