Chemical Modification of Papain and Subtilisin: An Active Site Comparison. An Undergraduate Biochemistry Experiment
Bibliographic record
Abstract
This experiment demonstrates the specific chemistry of cysteine and serine residues in the active sites of papain and subtilisin. While both protease enzymes catalyze the same reaction on similar substrates, papain uses cysteine to cleave peptide bonds and subtilisin employs serine for the same transformation. Treatment of both enzymes with methyl methanethiosulfonate and phenylmethylsulfonyl fluoride modifies papain and subtilisin, respectively, rendering them inactive. Methyl methanethiosulfonate reacts rapidly and exclusively with available thiols to form mixed disulfides. In the case of papain, the sole available thiol is cysteine-25 in the active site. Once this residue is modified, no further enzymatic activity is observed. Interestingly, the free thiol at cysteine-25 can be easily regenerated from the mixed disulfide, and the return of efficient catalysis can be observed. Phenylmethylsulfonyl fluoride reacts irreversibly with activated serines resulting in near complete inhibition of serine proteases. Subtilisin is modified at serine-221 to give the sulfonic ester rendering the enzyme ineffective at forming an acyl–enzyme complex with a substrate. The comparison of papain and subtilisin highlights the active site differences between the two proteases. The entire experiment can be completed in two, three-hour laboratory periods, or it could be shortened to a single, three-hour period.
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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".