The active site of β-galactosidase catalyzes the hydrolysis of its disaccharide substrate via "shallow" and "deep" binding. Monovalent potassium ions (K+) as well as divalent magnesium ions (Mg2+) are required for the enzyme's optimal activity. The beta-linkage of the substrate is cleaved by a terminal carboxyl group on the side chain of a glutamic acid.
In E. coli, Glu-461 was thought to be the nucleophile in the substitution reaction.However, it is now known that Glu-461 is an acid catalyst. Instead, Glu-537 is the actual nucleophile,[12] binding to a galactosyl intermediate.
In humans, the nucleophile of the hydrolysis reaction is Glu-268.