Transition State Analysis of the Aldol-Like Reaction Catalyzed by NeuB, a Sialic Acid Synthase, Reveals a Stepwise Cationic Mechanism
Bibliographic record
Abstract
High Resolution Image Download MS PowerPoint Slide The sialic acid synthase NeuB, and other α-carboxyketose synthases, are long-standing targets for inhibition by potential antimicrobial compounds. NeuB catalyzes an aldol-like reaction of phosphoenolpyruvate (PEP) and N -acetylmannosamine (ManNAc) that passes through a tetrahedral intermediate (THI) to form N -acetylneuraminic acid. We measured multiple kinetic isotope effects (KIEs) in order to determine the transition state (TS) structure of the NeuB-catalyzed reaction. As part of this study, the use of incomplete T 1 relaxation during KIE measurement by NMR was investigated, and then used to accelerate KIE measurements 9-fold. KIEs measured at the 3- 13 C, 2- 13 C and 2- 18 O positions in PEP reveal a stepwise mechanism of THI formation in which the PEP C3 to ManNAc C1′(aldehyde) bond (C3···C1′) is formed first, leading to a cationic intermediate. However, the transition state is so early in C3···C1′ bond formation, with a C3···C1′ bond order of 0.17 to 0.35, that there is little cationic character in the transition state. The lack of change in geometry or charge in forming the transition state implies that NeuB may function primarily by “catalysis-by-approximation”; i.e., its main contribution to TS stabilization is bringing the reactants together in the correct orientation. It also implies that inhibitors designed to mimic a positive charge are unlikely to be effective. Instead, neutral inhibitors that maximize the number of active site interactions are more likely to be effective.
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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 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.000 |
| Bibliometrics | 0.000 | 0.001 |
| 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".