Mechanistic Requirements for the Efficient Enzyme-Catalyzed Hydrolysis of Thiosialosides
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Bibliographic record
Abstract
The sialidase from Micromonospora viridifaciens has been found to catalyze the hydrolysis of aryl 2-thio-alpha-D-sialosides with remarkable efficiency: the first- and second-order rate constants, kcat and kcat/Km, for the enzyme-catalyzed hydrolysis of PNP-S-NeuAc are 196 +/- 5 s(-1) and (6.7 +/- 0.7) x 10(5) M(-1) s(-1), respectively. A reagent panel of eight aryl 2-thio-alpha-D-sialosides was synthesized and used to probe the mechanism for the M. viridifaciens sialidase-catalyzed hydrolysis reaction. In the case of the wild-type enzyme, the derived Brønsted parameters (beta(lg)) on kcat and kcat/Km are -0.83 +/- 0.11 and -1.27 +/- 0.17 for substrates with thiophenoxide leaving groups of pKa values > or = 4.5. For the general-acid mutant, D92G, the derived beta(lg) value on kcat for the same set of leaving groups is -0.82 +/- 0.12. When the conjugate acid of the departing thiophenol was < or = 4.5, the derived Brønsted slopes for both the wild-type and the D92G mutant sialidase were close to zero. In contrast, the nucleophilic mutant, Y370G, did not display a similar break in the Brønsted plots, and the corresponding values for beta(lg), for the three most reactive aryl 2-thiosialosides, on kcat and kcat/Km are -0.76 +/- 0.28 and -0.84 +/- 0.04, respectively. Thus, for the Y370G enzyme glycosidic C-S bond cleavage is rate-determining for both kcat and kcat/Km, whereas, for both the wild-type and D92G mutant enzymes, the presented data are consistent with a change in rate-determining step from glycosidic C-S bond cleavage for substrates in which the pKa of the conjugate acid of the leaving group is > or = 4.5, to either deglycosylation (kcat) or a conformational change that occurs prior to C-S bond cleavage (kcat/Km) for the most activated leaving groups. Thus, the enzyme-catalyzed hydrolysis of 2-thiosialosides is strongly catalyzed by the nucleophilic tyrosine residue, yet the C-S bond cleavage does not require the conserved aspartic acid residue (D92) to act as a general-acid catalyst.
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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 it