Mechanistic investigations of enzyme-catalyzed peptide and carbohydrate epimerization
Bibliographic record
Abstract
The inversion of stereochemistry in biomolecules is catalyzed by enzymes called racemases and epimerases. One such epimerase, isolated from the venom of the funnel-web spider Agelenopsis aperta, interconverts two peptide neurotoxins that differ only by the absolute stereochemistry at Ser46. Because enzymatic epimerization of peptides is otherwise unknown, the mechanism by which this occurs was investigated. Various substrate analogues were synthesized, in which the functional groups about the epimerizable serine were modified. One of these, containing chlorine in place of the hydroxyl, was found to undergo enzyme-catalyzed elimination of HC1 to generate a dehydroalanine derivative. This dehydroalanine peptide was independently synthesized and found to be a potent inhibitor of the epimerase, exhibiting a submicromolar IC₅₀. These results support a deprotonation-reprotonation mechanism that proceeds through an enolate intermediate. The planarity of this intermediate is apparently mimicked by the sp² character of the a-carbon of dehydroalanine, resulting in the observed inhibition. The biosynthesis of sialic acids is initiated by the conversion of UDP-N- acetylglucosamine (UDP-GlcNAc) to N-acetylmannosamine (ManNAc), a process involving epimerization at C-2. Whereas a hydrolyzing UDP-GlcNAc 2-epimerase is known to catalyze this reaction in mammals, a similar enzyme in bacteria had not been previously identified. The gene product from neuC, part of the Escherichia coli Kl gene cluster responsible for sialic acid synthesis, was shown by ¹H- and ³¹P-NMR spectroscopy and mass spectrometry to convert UDPGlcNAc to UDP and 2-acetamidoglucal. The homologous gene from Neisseria meningitidis B, siaA, was cloned and expressed as a histidine-tagged fusion protein. This protein, previously identified erroneously in literature in its untagged form as an N-acetylglucosamine-6-phosphate 2-epimerase, was shown to be a hydrolyzing UDP-GlcNAc 2-epimerase, converting UDPGlcNAc to α-ManNAc and UDP. Similar to the non-hydrolyzing UDP-GlcNAc 2-epimerase, SiaA was found to be allosterically regulated by its substrate, exhibiting sigmoidal kinetics with K[sub cat] = 4.7 s-¹, K¹ = 1.5 mM, and Hill coefficient, n [sub app] = 1.9. Additionally, in the presence of UDP, 2-acetamidoglucal was found to be a substrate, thereby implicating it as an intermediate. Incubations with (2"-²H)UDP-GlcNAc showed no significant kinetic isotope effect on kcat and kcJKm, indicating that deprotonation at C-2" is not rate limiting. A solvent isotope discrimination experiment, wherein either UDP-GlcNAc or 2-acetamidoglucal and UDP were incubated with the enzyme in 50% deuterated water, failed to show a preference for proton transfer over deuteron transfer during glycal hydration, indicating that this step is also not rate limiting. Finally, the ¹⁸O label from the incubation of [l"-¹⁸O]UDP-GlcNAc with SiaA was shown to depart with UDP, confirming that the reaction proceeds with C-O bond cleavage.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 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 | 0.001 |
| Open science | 0.001 | 0.000 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
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 source (direct Gemma or distilled Codex), 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".