Ground-state, transition-state, and metal-cation effects of the 2-hydroxyl group on β-D-galactopyranosyl transfer catalyzed by β-galactosidase (Escherichia coli, lac Z)

Substitution of the C2-OH group by C2-H at 4-nitrophenyl-β-D- galactopyranoside to give 4-nitrophenyl-2-deoxy-β-D-galactopyranoside causes (1) a change in the rate-determining step for β-galactosidase- catalyzed sugar hydrolysis from formation to breakdown of a covalent intermediate; (2) a 14 000-fold decrease in the second-order rate constant k₃/K_d for enzyme-catalyzed transfer of the β-D-galactopyranosyl group from the substrate to form a covalent adduct to the enzyme; and (3) a larger 320 000-fold decrease in the first-order rate constant k_s for hydrolysis of this covalent adduct. Only a small fraction (ca. 7%) of the 2-OH substituent effect is expressed in the ground-state Michaelis complex, so that the (apparent) strong interactions between the enzyme and 2-OH group that stabilize the transition state for β-D-galactopyranosyl transfer only develop upon moving from the Michaelis complex to the transition state. Mg²⁺ activates β-galactosidase for cleavage of both 4-nitrophenyl-β-D-galactopyranoside and 4-nitrophenyl-2-deoxy-β-D-galactopyranoside. This suggests that Mg ²⁺ activation does not involve interactions with the 2-OH group. The removal of Mg²⁺ from β-galactosidase causes a change in the rate-determining step for enzyme-catalyzed hydrolysis of 4-nitrophenyl-2-deoxy- β-D-galactopyranoside from breakdown to formation of the covalent intermediate. The observed 2-OH effect would require a very large (10-11 kcal/mol) stabilization of the transition state for β-D-galactopyranosyl group transfer to water by interactions between β-galactosidase and the neutral 2-OH group. We suggest that the apparent effect of the neutral substituent is more simply rationalized by ionization of the 2-OH to form a 2-O^- anion, which provides effective electrostatic stabilization of the cationic transition state for glycoside cleavage at an active site of relatively low dielectric constant.