Structure-Reactivity Relationships for β-Galactosidase (Escherichia coli, lac Z). 2. Reactions of the Galactosyl-Enzyme Intermediate with Alcohols and Azide Ion

Velocities for the synthesis of β-D-galactopyranosyl derivatives by transfer of the galactosyl group from β-galactosidase to seven alkyl alcohols, glucose, and azide ion have been determined as the difference in the velocities for β-galactosidase-catalyzed cleavage of 4-nitrophenyl β-D-galactopyranoside to give 4-nitrophenoxide anion (vpnp) and hydrolysis of this compound to give D-galactose (V_Gal). Rate constant ratios k_ROH/k_s (M^-1) for partitioning of the galactosylated enzyme between reaction with alkyl alcohols and solvent determined by this method are in good agreement with values of k_ROH/k_s (M^-1) determined by analysis of alcohol inhibition of enzyme-catalyzed hydrolysis of the corresponding alkyl β-D-galactopyranosides. Absolute rate constants k_ROH (M^-1 s^-1) for reaction of alkyl alcohols with the galactosylated enzyme intermediate were calculated from the corresponding rate constant ratio k_ROH/k_s (M^-1) and k_s = 710 s^-1. A Brpnsted parameter of (β_nuc)_ROH = -0.19 ± 0.10 was determined from the second-order rate constants for the reactions of alcohols with the galactosylated enzyme. The large difference between (β_1g)_kcat/Km = -0.75 ± 0.14 for cleavage of alkyl β-D-galactopyranosides to form the galactosylated enzyme and (β_nuc)_ROH = -0.19 for the reverse synthesis reaction requires that the equilibrium constants for galactosyl group transfer from alkyl β-D-galactopyranosides to the enzyme increase sharply with decreasing pk_a of the alkyl alcohol leaving group. These data give β_eq = -0.56 ± 0.05 for the reaction of alkyl β-D-galactopyranosides with ethanol to form ethyl β-D-galactopyranoside and alkyl alcohol. Several effects that lead to this increased ease of cleavage of alkyl β-D-galactopyranosides with decreasing basicity of the alkoxy group are discussed. A second-order rate constant of k_Glc = 1.2 x 10₄ M_-1 s_-1 was determined for reaction of glucose with the galactosylated enzyme. The relatively low reactivity of glucose is surprising, because an earlier observation that the galactosylated enzyme complex generated by the cleavage of lactose undergoes release of glucose and synthesis of allolactose at nearly equal rates suggests that the binding of glucose to the galactosylated enzyme should be partly irreversible and that it takes place near the encounter-controlled limit. The data suggest a significant stabilization of nonproductive complexes formed by binding of glucose to the galactosylated enzyme. β-Galactosidase catalyzes the hydrolysis of β-D-galactopyranosyl azide, but not the synthesis of this compound by reaction of azide ion with the galactosylated enzyme. This suggests that different forms of β-galactosidase catalyze the cleavage and synthesis of β-D-galactopyranosyl azide. This may correspond to a change in the state of ionization of the residue that participates in acid-base catalysis of the reaction in the cleavage and synthesis directions.