Notice bibliographique
Résumé
Flavin-containing oxidases are a class of proteins which use oxygen to regenerate the oxidized state of the isoalloxazine ring in flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) after it has been reduced by substrate oxidation. Though well characterized experimentally, many questions linger regarding how small molecules access the active site as well as where oxygen activation occurs in flavin-containing oxidases. A prototypical member of this family is monomeric sarcosine oxidase (MSOX), and it is perhaps the most well studied. Despite knowing which features are essential for catalysis as well as the location of the substrate binding site, it is unclear how oxygen accesses the site since the only clear entryway can be partially blocked by the larger substrate sarcosine. As such, two competing mechanisms have gained attention centering around the order by which ligands enter the binding site. In this thesis, we detail the use of all atom molecular dynamics (MD) studies to identify how oxygen accesses the MSOX active site, as well as characterize the resulting kinetic network. We use the single sweep method to identify four potential routes for oxygen to travel from the surface of MSOX to the active site. Then, using Markovian milestoning with Voronoi tessellations (MMVT), we refine the pathways identified in single sweep and develop a Markov state model describing the kinetics of oxygen entry and exit. We calculate entry and exit mean first passage times (MFPT) for oxygen from this model, which are used to compute second order rate constants for entry and first order rate constants for exit. Our calculated rate constants and mechanisms show that the presence of a substrate-mimicking inhibitor markedly influences the kinetics of oxygen entry and exit. The bound competitive inhibitor changes the protein structure sufficiently to shut down almost all major oxygen channels save one, which it opens, speeding entry but greatly slowing down oxygen exit, relative to the substrate-free enzyme. This means that our kinetic analysis predicts oxygen exhibits a longer residence time within MSOX when a substrate-like ligand is present. This supports the so-called "modified ping-pong" mechanism, in agreement with previous experimental results, thus lending validity to our approach. Furthermore, our computed second-order entry rate constants are larger by about an order of magnitude than are experimentally determined oxygen consumption rate constants. Since oxygen consumption combines the processes of entry and electron transfer, we conclude that of these two, entry is not rate-limiting in the overall catalytic cycle, regardless of whether or not a substrate-like ligand is bound. Finally, because this work represents the first test of the MMVT approach for comparing kinetics of ligand entry for an enzyme in two distinct states, we not surprisingly uncovered inefficiencies in the approach. We tested one idea for gaining efficiency based on the "finite-temperature" string method, in which transport channels can be determined and kinetically characterized on-the-fly, rather than sequentially. Our results indicate that more research in that area is needed.
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 | 0,000 | 0,001 |
| Méta-épidémiologie (sens strict) | 0,000 | 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 | 0,001 | 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) | 0,001 | 0,000 |
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 ».