Recruitment of Both Uniform and Differential Binding Energy in Enzymatic Catalysis: Xylanases from Families 10 and 11
Bibliographic record
Abstract
The contributions of enzyme-substrate hydrogen-binding interactions to catalysis by two different families of xylanases were evaluated through kinetic studies with two representative wild-type enzymes, Cellulomonas fimi xylanase (Cex) and Bacillus circulans xylanase (Bcx), on a series of monodeoxygenated and monodeoxyfluorinated p-nitrophenyl xylobioside substrates. Effects of substitution in the distal (-2 subsite) sugar on kcat/Km for Cex were moderately large (up to 2.9 kcal mol-1), with no effect seen on kcat. By contrast, substantial effects upon both kcat and kcat/Km were seen for substrates modified in the proximal (-1 subsite) sugar. Very similar results were obtained with Bcx. Kinetic analyses with a series of eight mutants of Cex in which active site residues interacting with the substrate were mutated yielded complementary insights. Again, interactions with the distal (-2) sugar were seen to contribute substantially to kcat/Km (up to 3.7 kcal mol-1), thus to the formation of the glycosyl-enzyme intermediate, but not to kcat, thus to the hydrolysis of the glycosyl-enzyme. Interactions with the proximal (-1) sugar are much more significant, contributing up to 6.7 kcal mol-1 to both kcat/Km and kcat. These results together indicate that interactions with the distal sugar maintain similar magnitudes in the transition states for glycosylation and deglycosylation as well as in the glycosyl-enzyme intermediate and can be referred to as "uniform binding interactions" in the parlance of Albery and Knowles (Albery, W. J., and Knowles, J. R. (1976) Biochemistry 15, 5631-5640). Interactions with the proximal sugar are considerably stronger at the deglycosylation transition state than in the intermediate, and fall into the category of differential binding interactions. This behavior likely has its origins in the changes in ring conformation of the proximal sugar but not of the distal sugar between the ground state and the reaction transition state. Correlation of these individual interaction energies with the hydrogen-bonding pattern seen in the glycosyl-enzyme intermediate allows for the assignment of hydrogen-bond strengths to each interaction, with good correlation between the two approaches. These findings are relevant to the discussion of remote binding effects upon enzymatic catalysis.
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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".