Title:Mutation Studies in the Active Site of β-glycosidase from Pyrococcus furiosus DSM 3638
Volume: 20
Issue: 1
Author(s): Sunghoon Park, Inteaz Alli, Kwanhwa Park, Byungchul Oh and Byonghoon Lee
Affiliation:
Keywords:
PFTG (Pyrococcus furiousus thermostable glycosidase), hyperthermostable enzyme, ITC (isothermal titration calorimetry), glycoside hydrolase, β-galactosidas, catalytic site, pH, enzyme
Abstract: Sequence alignments and homology modeling of Pyrococcus furiosus thermostable glycosidase (PFTG)
showed that the residue 150 is conserved as tryptophan in β-glycosidase and in other related enzymes such as β-
mannosidase and β-galactosidase. To elucidate the relationship between the substrate size and geometric shape of the
catalytic site of thermophilic β-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°C
and pH 5.0 in sodium citrate buffer, respectively. The catalytic efficiencies (kcat/Km) of the mutants on synthetic and natural
substrates by Isothermal Titration Calorimetry were slightly changed, but indicated the characteristics of β-glycosidase
activity. Kinetic parameters of the mutant enzymes indicated that they possess characteristics of both β- galactosidase and
β-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 (kcat).
