Mechanistic investigations of enzyme-catalyzed peptide and carbohydrate epimerization
Notice bibliographique
Résumé
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.
Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.
Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,001 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,001 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,001 |
| Science ouverte | 0,001 | 0,000 |
| Intégrité de la recherche | 0,001 | 0,001 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,002 | 0,001 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».