Intrinsic Nucleophilicity of Inverting and Retaining Glycoside Hydrolases Revealed Using Carbasugar Glyco-Tools
Bibliographic record
Abstract
Hydrolyses of cyclohexenyl-based carbasugars that mimic either α-d-glucose or α-d-galactose were explored with two Bacteroides thetaiotaomicron enzymes from glycoside hydrolase family 97: an inverting α-glucosidase (BtGH97a) and a retaining α-galactosidase (BtGH97b). Both enzymes yield nucleophilic substitutions at the pseudo-anomeric center of the carbasugar substrates, giving significantly different linear energy relationships for the catalytic rate constant as a function of the leaving group ability. Specifically, the kinetic data for the inverting α-glucosidase is consistent with the reaction giving a hydrolyzed inverted carbaglucose product by a mechanism that proceeds with little nucleophilic participation by the bound water molecule at the reaction transition state. In contrast, the reaction of carbagalactose substrates with the retaining GH97 enzyme involves a rate-limiting nonchemical step, likely a conformational change, followed by rapid substitution involving a nucleophilic amino acid residue to give a covalently bound intermediate. Considering the structural similarities between these two GH97 enzymes, the kinetic data nonetheless reveal a significant (>106) difference in the rates of nucleophilic attack between the unique enzymatic nucleophiles─with the less nucleophilic species being H2O in the inverting α-glucosidase and the better nucleophile being a carboxylate in the retaining α-galactosidase. The enzymatic rate constant ratio for the phenyl carbasugars contrasts with the corresponding kinetic data obtained using natural substrate phenyl glycopyranosides. Last, for the galactocarbasugar with a phenol leaving group, the second-order rate constant for alkylation of the GH97 α-galactosidase is only ∼10-fold lower than that for glycosylation of this enzyme by the parent carbohydrate phenyl α-d-galactopyranoside. This modest difference in rate constants underscores our conclusion that retaining glycoside hydrolases may not have optimized the nucleophilicity of their active site nucleophiles with the result that the transition state free energies for formation and hydrolysis of the covalent enzyme intermediate are matched.
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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 | 0.001 |
| 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".