Kinetic Characterization and Identification of the Acylation and Glycosylation Sites of Recombinant Human γ-Glutamyltranspeptidase
Bibliographic record
Abstract
Gamma-glutamyltranspeptidase (GGT) is a heterodimeric enzyme important for glutathione homeostasis control. It has also been implicated in many physiological disorders, including Parkinson's disease, apoptosis inhibition, and diabetes. In the first step of its ping-pong mechanism it binds glutathione, its in vivo substrate, and releases cysteinylglycine upon formation of an acyl-enzyme intermediate. This intermediate can then react with water to release glutamate as a hydrolysis product or with an amino acid or dipeptide to form a transpeptidation product. Further detailed study of the mechanism underlying these reactions is hindered at least for some GGTs by the low quantities of protein available after a multistep purification from tissue. In the present work the gene for human GGT was cloned into the pPICZalphaA vector and transformed into Pichia pastoris to express as a 68 kDa His-tagged protein. The optimized expression and secretion of this enzyme in 1 L of culture and subsequent purification by immobilized metal affinity chromatography yielded 1.6 mg of purified enzyme having a specific activity of 237 U/mg. Kinetic parameters for the transpeptidation reaction between glutathione and glycylglycine were determined by mass spectrometry, giving a kcat of 13.4 x 10(3) min-1 and apparent KM values of 1.11 mM for glutathione and 8.1 mM for glycylglycine. The GGT-mediated hydrolysis of glutathione was also studied, providing a kcat of 53 min-1 and a KM value of 7.3 microM for glutathione. Incubation of the enzyme with a mechanism-based inhibitor, enzymatic digest, and mass spectrometric analysis provided the first unambiguous identification of Thr381 as the active site nucleophile of human gamma-glutamyltranspeptidase, and confirmed four of the seven N-linked glycosylation sites. These structural and kinetic data are discussed with respect to a homology model generated to facilitate visualization.
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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".