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 pr imary alcohols, phenol and azide has been studied during the reactions at 8 0 degreesC of thermostable beta -glycosidases from Sulfolobus solfataricus (Ss beta Gly) and Pyrococcus furiosus (CelB) with 2-nitrophenyl beta -D-gal actopyranoside 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 th e difference in the initial velocities of the formation of 2-nitrophenol or D-glucose, and D-galactose. The results show that hydrophobic bonding inte ractions contribute approximate to8 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-o ctanol. Values of k(Nu)/k(water) for reactions of galactosylated Ss beta Gl y with ethanol and substituted derivatives of ethanol show no clear depende nce 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 nonp roductive pertaining to beta -galactoside synthesis. Neither enzyme catalyz es galactosyl transfer to azide ion. A model is proposed for the interactio n of neutral nucleophiles at an extended acceptor site of the galactosylate d en-zymes.