Site-saturation Mutagenesis of Tyr-105 Reveals Its Importance in Substrate Stabilization and Discrimination in TEM-1 β-Lactamase
Bibliographic record
Abstract
The conserved Class A beta-lactamase active site residue Tyr-105 was substituted by saturation mutagenesis in TEM-1 beta-lactamase from Escherichia coli in order to clarify its role in enzyme activity and in substrate stabilization and discrimination. Minimum inhibitory concentrations were calculated for E. coli cells harboring each Y105X mutant in the presence of various penicillin and cephalosporin antibiotics. We found that only aromatic residues as well as asparagine replacements conferred high in vivo survival rates for all substrates tested. At position 105, the small residues alanine and glycine provide weak substrate discrimination as evidenced by the difference in benzylpenicillin hydrolysis relative to cephalothin, two typical penicillin and cephalosporin antibiotics. Kinetic analyses of mutants of interest revealed that the Y105X replacements have a greater effect on K(m) than k(cat), highlighting the importance of Tyr-105 in substrate recognition. Finally, by performing a short molecular dynamics study on a restricted set of Y105X mutants of TEM-1, we found that the strong aromatic bias observed at position 105 in Class A beta-lactamases is primarily defined by a structural requirement, selecting planar residues that form a stabilizing wall to the active site. The adopted conformation of residue 105 prevents detrimental steric interactions with the substrate molecule in the active site cavity and provides a rationalization for the strong aromatic bias found in nature at this position among Class A beta-lactamases.
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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 | 0.000 | 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) | 0.001 | 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 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".