Transgalactosylation by thermostable beta-glycosidases from Pyrococcus furiosus and Sulfolobus solfataricus - Binding interactions of nucleophiles with the galactosylated enzyme intermediate make major contributions to the formation of new beta-glycosides during lactose conversion

The hyperthermostable beta-glycosidases from the Archaea Sulfolobus solfata ricus (Ss beta Gly) and Pyrococcus furiosus (CelB) hydrolyse beta-glycoside s of D-glucose or D-galactose with relaxed specificities pertaining to the nature of the leaving group and the glycosidic linkage. To determine how sp ecificity is manifested under conditions of kinetically controlled transgal actosylation, the major transfer products formed during the hydrolysis of l actose by these enzymes have been identified, and their appearance and degr adation have been determined in dependence of the degree of substrate conve rsion. CelB and Ss beta Gly show a marked preference for making new beta(1- ->3) and beta(1-->6) glycosidic bonds by intermolecular as well as intramol ecular transfer reactions. The intramolecular galactosyl transfer of CelB, relative to glycosidic-bond cleavage and release of glucose, is about 2.2 t imes that of Ss beta Gly and yields beta-D-Galp-(1-->6)-D-Glc and beta-D-Ga lp-(1-->3)-D-Glc in a molar ratio of approximate to 1 : 2. The partitioning of galactosylated Ss beta Gly between reaction with sugars [k(Nu) (M-1.S-1 )] and reaction with water [k(water) (s(-1))] is about twice that of CelB. It gives a mixture of linear P-D-glycosides, chiefly trisaccharides at earl y reaction times, in which the prevailing new glycosidic bonds are beta(1-- >6) and beta(1-->3) for the reactions catalysed by Ss beta Gly and CelB, re spectively. The accumulation of beta-D-Galp-(1-->6)-D-Glc at the end of lac tose hydrolysis reflects a 3-10-fold specificity of both enzymes for the hy drolysis of beta(1-->3) over beta(1-->6) linked glucosides. Galactosyl tran sfer from Ss beta Gly or CelB to D-glucose occurs with partitioning ratios, k(Nu)/k(water), which are seven and >170 times those for the reactions of the galactosylated enzymes with I-propanol and 2-propanol, respectively. Th erefore, the binding interactions with nucleophiles contribute chiefly to f ormation of new beta-glycosides during lactose conversion. Likewise, noncov alent interactions with the glucose leaving group govern the catalytic effi ciencies for the hydrolysis of lactose by both enzymes. They are almost ful ly expressed in the rate-limiting first-order rate constant for the galacto syl transfer from the substrate to the enzyme and lead to a positive deviat ion by approximate to 2.5 log(10) units from structure-reactivity correlati ons based on the pK(a) of the leaving group.