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Updated: Jul 18, 2026

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis
Published on: March 17, 2010
Computational Insights into Cyst(e)inase for Cysteine and Cystine Depletion Therapies: Free Energy Calculations,
Qian Zhang1, Xudong Lü1,2, Yuanyuan Jin1
1NHC Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China.
Abstract:
Cyst(e)inase, an engineered human cystathionine-γ-lyase (hCGL), effectively degrades l-Cys and CSSC, inhibiting various tumors in vivo with minimal toxicity. However, its current activity and stability limit its clinical application. To enhance its therapeutic potential, we utilized folding free energy calculations and amino acid site conservation to identify 46 potential mutants. Among them, 17 mutants exhibited improved activity or stability, while six showed a loss of function. Notably, mutant L142E enhanced activity for both substrates, and E349S displayed 2.8 times higher activity toward CSSC and a 12.8 °C increase in Tm, despite reduced activity toward l-Cys. Saturation mutagenesis highlighted the importance of spatial constraints for substrate selectivity. MD simulations suggested that distal mutations stabilize the near-attack conformation, enhancing the activity. In vitro cytotoxicity assays confirmed that the mutants L142E and E349S significantly inhibited gastric and pancreatic cancer cells. This study provides valuable insights for improving therapeutic enzyme design.
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