STRUCTURE-REACTIVITY RELATIONSHIPS FOR BETA-GALACTOSIDASE (ESCHERICHIA-COLI, LAC-Z) .3. EVIDENCE THAT GLU-461 PARTICIPATES IN BRONSTED ACID-BASE CATALYSIS OF BETA-D-GALACTOPYRANOSYL GROUP-TRANSFER

Experiments are reported to determine the role of Glu-461 in the beta- D-galactopyranosyl group transfer reaction catalyzed by beta-galactosi dase. E461G beta-galactosidase catalyzes the hydrolysis of 4-nitrophen yl beta-D-galactopyranoside through a galactosyl-enzyme intermediate t hat shows a high reactivity toward the anionic nucleophile azide ion, but no detectable reactivity toward the neutral nucleophile trifluoroe thanol, BY contrast, the galactosylated wildtype enzyme is reactive to ward trifluoroethanol but not anions. The change in specificity observ ed for the E461G mutant can be rationalized by a mechanism in which Gl u-461 participates in general acid-base catalysis at the leaving group /nucleophile. The observed low activity of E461G beta-galactosidase fo r hydrolysis of 2,2,2-trifluoroethyl beta-D-galactopyranoside is due e ntirely to a wild type enzyme contaminant in our preparation of the mu tant enzyme, and the mutant enzyme itself has essentially no catalytic activity for cleavage of this substrate. The substitution of glutamat e at position 461 by glycine leads to a more than 500 000-fold reducti on in the rate constant for enzymatic cleavage of the glycosidic bond to the strongly basic trifluoroethoxide leaving group (pK(a) = 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 ( pK(a) = 7.1). This corresponds to a more than 3.5 kcal/mol greater sta bilization by Glu-461 of the transition state for the reaction of the substrate with the more basic trifluoroethoxide leaving group. These d ata are consistent with the conclusion that Glu-461 provides general a cid catalysis of leaving group departure, which is most effective for cleavage of the relatively strong bonds to basic alkoxide leaving grou ps.