STRUCTURE-REACTIVITY RELATIONSHIPS FOR BETA-GLACTOSIDASE (ESCHERICHIA-COLI, LAC Z) .4. MECHANISM FOR REACTION OF NUCLEOPHILES WITH THE GALACTOSYL-ENZYME INTERMEDIATES OF E461G AND E461Q BETA-GALACTOSIDASES

Second-order rate constants for transfer of the beta-D-galactopyranosy l group from the galactosyl-enzyme intermediates of the galactosyl tra nsfer reactions catalyzed by E461G and E461Q beta-galactosidases to an ionic nucleophiles have been determined. The second-order rate constan t for reaction of the galactosylated E461G enzyme with azide ion is 49 00 M(-1) s(-1). By contrast, there is no detectable reaction of the ga lactosylated wild type enzyme with azide ion (Richard et al., 1995b), and the E461G mutation leads to a large decrease in the second-order r ate constant k(cat)/K-m for catalysis of cleavage of beta-D-galactopyr anosyl azide, which is the microscopic reverse of the reaction of azid e ion with the galactosyl-enzyme intermediate. These data show that th e E461G mutation causes a more than 8000-fold increase in the equilibr ium constant for transfer of the beta-D-galactopyranosyl group from be ta-galactosidase to azide ion. We propose that this change represents the requirement for the coupling of galactosyl transfer from the nativ e enzyme to the thermodynamically unfavorable protonation of the carbo xylate group of Glu-461, but the expression of the full chemical affin ity of azide ion for galactosyl transfer from the mutant enzyme which lacks this ionizable side chain at position 461. The reactions of acet ate, butyrate and methoxyacetate ions with the galactosylated E461G en zyme and of acetate with the galactosylated E461Q enzyme give both the corresponding beta-galactopyranosyl derivatives and D-galactose, and the formation of the latter represents formal catalysis of the reactio n of water with the galactosylated enzyme. However, the reaction of fo rmate ion with the galactosylated E461G enzyme gives only D-galactose. These results suggest that carboxylate anions can take the place of t he excised propionate side chain of Glu-461 to provide general base ca talysis of the reaction of water with the galactosyl-enzyme intermedia tes. The relative reactivity of anionic nucleophiles toward the covale nt galactosyl-enzyme intermediate of the reactions catalyzed by the E4 61G enzyme is similar to that observed for partitioning of stable carb ocations in water, This suggests that replacement of the anionic side chain of Glu-461 by a hydrogen exposes an enzyme-stabilized oxocarbeni um ion intermediate to reaction with external nucleophilic reagents.