Mutation Studies in the Active Site of β-glycosidase from Pyrococcus furiosus DSM 3638
Bibliographic record
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). Keywords: PFTG (Pyrococcus furiousus thermostable glycosidase), hyperthermostable enzyme, ITC (isothermal titration calorimetry), glycoside hydrolase, β-galactosidas, catalytic site, pH, enzyme
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".