Biochemical and Functional Characterization by Site-Directed Mutagenesis of a Phospholipase A2 from Scorpio maurus Venom
Bibliographic record
Abstract
The study of amino acid interactions in the active site of scorpion venom phospholipases A2 could help to gain insights into the structure–function relationship and the biological activities of the enzyme. In the secreted phospholipase A2 of Scorpio maurus venom glands, Glutamate 63 and Tyrosine 122 amino acids play critical roles in the catalytic mechanism through interactions with residues around the calcium-binding loop. We constructed mutants at these positions by overexpression in Escherichia coli cells. After refolding and purification of recombinant enzymes, we studied their kinetic properties using pH-stat and monolayer techniques. The mutant Glutamate 63–Aspartate (E63D) exhibited a reduced activity, while the second mutant Tyrosine 122–Arginine (Y122R) retained some activity with a 14-fold reduction in catalytic efficiency. However, both mutants remained stable in pH values ranging from 2 to 12 whereas the double mutant D63–R122 was catalytically inactive. Comparative analysis of wild-type and mutant 3-D models showed various modifications of the hydrogen-binding network linking residues Glutamate 63 and Tyrosine 122. These modifications of interactions could explain the reduction in enzymatic activity. The kinetic behavior on phosphatidylcholine and phosphatidylethanolamine monolayers of three mutants was evaluated using a baro-stat system to assess the potential association between the hydrolysis of erythrocyte membrane phospholipids and the enzyme’s capability to penetrate phospholipid monolayers at high surface pressure. Mutants’ kinetic behaviors were similar to the wild-type form with slightly modified specific activities at high surface pressure. All mutants were more active on phosphatidylethanolamine than phosphatidylcholine films at high surface pressure. This study provided new information to further elucidate structure–function relationships of scorpion venom-secreted phospholipases A2 and the design of novel potent drug molecules.
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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) | 0.000 | 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) | 0.000 | 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".