Galactosyl transfer catalyzed by thermostable beta-glycosidases from Sulfolobus solfataricus and Pyrococcus furiosus: kinetic studies of the reactions of galactosylated enzyme intermediates with a range of nucleophiles.

The transfer of a galactosyl group from an enzyme to a number of neutral primary alcohols, phenol and azide has been studied during the reactions at 80 degrees C of thermostable beta-glycosidases from Sulfolobus solfataricus (Ss beta Gly) and Pyrococcus furiosus (CelB) with 2-nitrophenyl beta-D-galactopyranoside or lactose (4-O-beta-D-galactopyranosyl D-glucopyranose) as substrates. The rate constant ratios, k(Nu)/k(water), for partitioning of the galactosylated enzyme intermediates between reaction with nucleophiles (k(Nu), M(-1) s(-1)) and water (k(water), s(-1)) have been determined from the difference in the initial velocities of the formation of 2-nitrophenol or D-glucose, and D-galactose. The results show that hydrophobic bonding interactions contribute approximately 8 kJ mol(-1) to the stabilization of the transition state for the reaction of galactosylated enzyme intermediates of Ss beta Gly and CelB with 1-butanol, compared to the transition state for the enzymatic reaction with methanol. The leaving group/nucleophile binding sites of Ss beta Gly and CelB appear about 0.8 times as hydrophobic as n-octanol. Values of k(Nu)/k(water) for reactions of galactosylated Ss beta Gly with ethanol and substituted derivatives of ethanol show no clear dependence on the pK(a) of the primary hydroxy group of these nucleophiles in the pK(a) range 12.4-16.0. The binding of phenol with the galactosylated enzyme intermediates of Ss beta Gly and CelB occurs in a form that is mainly nonproductive pertaining to beta-galactoside synthesis. Neither enzyme catalyzes galactosyl transfer to azide ion. A model is proposed for the interaction of neutral nucleophiles at an extended acceptor site of the galactosylated enzymes.