Mutational Evidence of Transition State Stabilization by Serine 88 in <i>Escherichia coli</i> Type I Signal Peptidase<sup>,</sup>
Bibliographic record
Abstract
Type I signal peptidase (SPase I) catalyzes the hydrolytic cleavage of the N-terminal signal peptide from translocated preproteins. SPase I belongs to a novel class of Ser proteases that utilize a Ser/Lys dyad catalytic mechanism instead of the classical Ser/His/Asp triad found in most Ser proteases. Recent X-ray crystallographic studies indicate that the backbone amide nitrogen of the catalytic Ser 90 and the hydroxyl side chain of Ser 88 might participate as H-bond donors in the transition-state oxyanion hole. In this work, contribution of the side-chain Ser 88 in Escherichia coli SPase I to the stabilization of the transition state was investigated through in vivo and in vitro characterizations of Ala-, Cys-, and Thr-substituted mutants. The S88T mutant maintains near-wild-type activity with the substrate pro-OmpA nuclease A. In contrast, substitution with Cys at position 88 results in more than a 740-fold reduction in activity (k(cat)) whereas S88A retains much less activity (>2440-fold decrease). Measurements of the kinetic constants of the individual mutant enzymes indicate that these decreases in activity are attributed mainly to decreases in k(cat) while effects on K(m) are minimal. Thermal inactivation and CD spectroscopic analyses indicate no global conformational perturbations of the Ser 88 mutants relative to the wild-type E. coli SPase I enzyme. These results provide strong evidence for the stabilization by Ser 88 of the oxyanion intermediate during catalysis by E. coli SPase I.
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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".