Mutation studies in the active site of β-glycosidase from Pyrococcus furiosus DSM 3638.
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 (k(cat)/K(m)) 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 (K(m)) to substrates with low turnover number (k(cat)).
Department of Food Science, McGill University, Montreal, Quebec, Canada.
Sunghoon
S
Park
Inteaz
I
Alli
Kwanhwa
K
Park
Byungchul
B
Oh
Byonghoon
B
Lee
D000595:Amino Acid Sequence; D002151:Calorimetry; D020134:Catalytic Domain / Q000235:genetics*; D005973:Glutamine / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D020027:Pyrococcus furiosus / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry; D001616:beta-Galactosidase / Q000235:genetics; D044902:beta-Mannosidase / Q000235:genetics
D005973:Glutamine; D014364:Tryptophan; D006026:Glycoside Hydrolases; D001616:beta-Galactosidase; D044902:beta-Mannosidase