Differential mechanism‐based labeling and unequivocal activity assignment of the two active sites of intestinal lactase/phlorizin hydrolase
Bibliographic record
Abstract
Milk lactose is hydrolysed to galactose and glucose in the small intestine of mammals by the lactase/phlorizin hydrolase complex (LPH; EC 3.2.1.108/62). The two enzymatic activities, lactase and phlorizin hydrolase, are located in the same polypeptide chain. According to sequence homology, mature LPH contains two different regions (III and IV), each of them homologous to family 1 glycosidases and each with a putative active site. There has been some discrepancy with regard to the assignment of enzymatic activity to the two active sites. Here we show differential reactivity of the two active sites with mechanism-based glycosidase inhibitors. When LPH is treated with 2',4'-dinitrophenyl 2-deoxy-2-fluoro-beta-D-glucopyranoside (1) and 2', 4'-dinitrophenyl-2-deoxy-2-fluoro-beta-D-galactopyranoside (2), known mechanism-based inhibitors of glycosidases, it is observed that compound 1 preferentially inactivates the phlorizin hydrolase activity whereas compound 2 is selective for the lactase active site. On the other hand, glycals (D-glucal and D-galactal) competitively inhibit lactase activity but not phlorizin hydrolase activity. This allows labeling of the phlorizin site with compound 1 by protection with a glycal. By differential labeling of each active site using 1 and 2 followed by proteolysis and MS analysis of the labeled fragments, we confirm that the phlorizin hydrolysis occurs mainly at the active site located at region III of LPH and that the active site located at region IV is responsible for the lactase activity. This assignment is coincident with that proposed from the results of recent active-site mutagenesis studies [Zecca, L., Mesonero, J.E., Stutz, A., Poiree, J.C., Giudicelli, J., Cursio, R., Gloor, S.M. & Semenza, G. (1998) FEBS Lett. 435, 225-228] and opposite to that based on data from early affinity labeling with conduritol B epoxide [Wacker, W., Keller, P., Falchetto, R., Legler, G. & Semenza, G. (1992) J. Biol. Chem. 267, 18744-18752].
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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".