Mutation Studies in the Active Site of beta-glycosidase from Pyrococcus furiosus DSM 3638

Abstract

Sequence alignments and homology modeling of Pyrococcus furiosus thermostable glycosidase (PFTG) showed that the residue 150 is conserved as tryptophan in beta-glycosidase and in other related enzymes such as mannosidase and beta-galactosidase. To elucidate the relationship between the substrate size and geometric shape of the catalytic site of thermophilic beta-glycosidase and category of PFTG, the Q77, Q150 and D206 located at the interface of the dimer were replaced with Trp and Asn. Also, to confirm the role of active sites of PFTG, the Q77R/Q150W double mutant was created through subcloning. Temperature and pH optima of both mutants and native enzyme were same at 100 degrees C and pH 5.0 in sodium citrate buffer, respectively. The catalytic efficiencies (k(cat)/K-m) of the mutants on synthetic and natural substrates by Isothermal Titration Calorimetry were slightly changed, but indicated the characteristics of beta-glycosidase activity. Kinetic parameters of the mutant enzymes indicated that they possess characteristics of both beta-galactosidase and beta-mannosidase activities. Although the mutant enzymes showed similar substrate specificities compared to the recombinant enzyme, they had more affinity (Km) to substrates with low turnover number (k(cat)).