INVESTIGATION OF THE CATALYTIC AND REGULATORY DOMAINS OF CYSTATHIONINE β-SYNTHASE
Bibliographic record
Abstract
Cysthationine β-Synthase (CBS) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the condensation of serine and homocysteine to form cysthathionine.Human CBS (hCBS) is a modular enzyme that contains an N-terminal domain that binds heme, a catalytic domain that binds the PLP cofactor and a regulatory domain that binds S-adenosyl-methionine (SAM).The interaction between the catalytic and regulatory regions is limited to a linker region that forms the main communication between these two domains.Deficiency in CBS activity is the leading cause for the disorder homocystinuria in humans.The focus of the studies described in this thesis is to investigate the mechanism of homocystinuria-associated mutations in the active site of yeast CBS, the potential of native tryptophan residues as probes of conformational change and the binding of SAM to the regulatory domain.Site-directed replacement variants of 9 residues located in the active-site of the truncated form of yeast CBS (residues 1-353) were characterized.The results suggest that the hydrogen bonding network comprising residues K112, E111, K327, E244 and T326 is a determinant of active-site architecture and dynamics.Residues G245, I246 and G247, situated adjacent to the cofactor, play a role in maintaining PLP in a catalytically productive orientation.With the goal of developing probes of conformational change within the catatlytic domain and of communication between the catalytic and regulatory domain of CBS, single and triple-substitution, site-directed variants of the four tryptophan residues of yeast CBS (W132, W263, W333 and W340) were characterized in the truncated and fulllength enzyme forms.Residues W132 and W263 are located in the active site, while iii W333 and W340 are located in the linker region that connects the catalytic and regulatory domains.The results demonstrate that residue W263 is the main contributor for fluorescence resonance energy transfer to the PLP cofactor.Substitution of W333 and W340 causes a change in the degree of solvent exposure in the microenvironment of these residues.This is due to the presence of the regulatory domain in the full-length enzyme.Binding of SAM to the regulatory domain of human CBS leads to enzyme activation.In contrast, the Drosophila melanogaster and yeast CBS enzymes are not activated by SAM.The binding of SAM to the regulatory domains of yeast and human CBS was investigated via fluorescence spectroscopy and mass spectrometry.The results indicate that the full-length yeast enzyme does not bind to SAM, suggesting that, similar to the drosophila enzyme, yeast CBS may locked in a conformation that does not allow SAM-binding to its regulatory domain.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| 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) | 0.001 | 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 source (direct Gemma or distilled Codex), 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".