Penicillopepsin‐JT2, a recombinant enzyme from <i>Penicillium janthinellum</i> and the contribution of a hydrogen bond in subsite S<sub>3</sub> to k<sub><i>cat</i></sub>
Bibliographic record
Abstract
Abstract The nucleotide sequence of the gene ( pepA ) of a zymogen of an aspartic proteinase from Penicillium janthinellum with a 71% identity in the deduced amino acid sequence to penicillopepsin (which we propose to call penicillopepsin‐JT1) has been determined. The gene consists of 60 codons for a putative leader sequence of 20 amino acid residues, a sequence of about 150 nucleotides that probably codes for an activation peptide and a sequence with two introns that codes for the active aspartic proteinase. This gene, inserted into the expression vector pGPT‐pyrG1, was expressed in an aspartic proteinase‐free strain of Aspergillus niger var. awamori in high yield as a glycosylated form of the active enzyme that we call penicillopepsin‐JT2. After removal of the carbohydrate component with endoglycosidase H, its relative molecular mass is between 33,700 and 34,000. Its kinetic properties, especially the rate‐enhancing effects of the presence of alanine residues in positions P 3 and P 2 ′ of substrates, are similar to those of penicillopepsin‐JT1, endothia‐pepsin, rhizopuspepsin, and pig pepsin. Earlier findings suggested that this rate‐enhancing effect was due to a hydrogen bond between the ‐NH‐of P 3 and the hydrogen bond accepting oxygen of the side chain of the fourth amino acid residue C‐terminal to Asp215. Thr219 of penicillopepsin‐JT2 was mutated to Ser, Val, Gly, and Ala. Thr219Ser showed an increase in k cat when a P 3 residue was present in the substrate, which was similar to that of the wild‐type, whereas the mutants Thr219Val, Thr219Gly, and Thr219Ala showed no significant increase when a P 3 residue was added. The results show that the putative hydrogen bond alone is responsible for the increase. We propose that by locking the ‐NH‐ of P 3 to the enzyme, the scissile peptide bond between P 1 and P 1 ′ becomes distorted toward a tetrahedral conformation and becomes more susceptible to nucleophilic attack by the catalytic apparatus without the need of a conformational change in the enzyme. Lyme borreliosis is a multisystem disorder caused by the spirochete Borrelia burgdorferi that is transmitted to humans by the tick Ixodes dammini. The immune response against the 31 kDa OspA, which is one of the most abundant B. burgdorferi proteins, appears to be critical in preventing infection and tissue inflammation. Detailed knowledge of the immunological and molecular characteristics of the OspA protein is important for the development of reliable diagnostic assays. In this study, we characterized a new conformational epitope present within the middle part of B. burgdorferi OspA. Our approach used enzymatic proteolyses of the immune complex followed by mass spectrometric identification of the peptides bound to the antibody. It appears to be one of the first reports on the characterization of a discontinuous epitope using mass spectrometry.
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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 | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.001 |
| Science and technology studies | 0.000 | 0.001 |
| 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".