Experiments are reported to determine the role of Glu-461 in the beta-D-galactopyranosyl group transfer reaction catalyzed by beta-galactosidase. E461G beta-galactosidase catalyzes the hydrolysis of 4-nitrophenyl beta-D-galactopyranoside through a galactosyl-enzyme intermediate that shows a high reactivity toward the anionic nucleophile azide ion, but no detectable reactivity toward the neutral nucleophile trifluoroethanol. By contrast, the galactosylated wild type enzyme is reactive toward trifluoroethanol but not anions. The change in specificity observed for the E461G mutant can be rationalized by a mechanism in which Glu-461 participates in general acid-base catalysis at the leaving group/nucleophile. The observed low activity of E461G beta-galactosidase for hydrolysis of 2,2,2-trifluoroethyl beta-D-galactopyranoside is due entirely to a wild type enzyme contaminant in our preparation of the mutant enzyme, and the mutant enzyme itself has essentially no catalytic activity for cleavage of this substrate. The substitution of glutamate at position 461 by glycine leads to a more than 500 000-fold reduction in the rate constant for enzymatic cleavage of the glycosidic bond to the strongly basic trifluoroethoxide leaving group (pKa = 12.4), but to a smaller 1300-fold reduction in the rate constant for cleavage of the bond to the more weakly basic 4-nitrophenoxide leaving group (pKa = 7.1). This corresponds to a more than 3.5 kcal/mol greater stabilization by Glu-461 of the transition state for the reaction of the substrate with the more basic trifluoroethoxide leaving group. These data are consistent with the conclusion that Glu-461 provides general acid catalysis of leaving group departure, which is most effective for cleavage of the relatively strong bonds to basic alkoxide leaving groups.