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Updated: Dec 24, 2025

Optimizing the Growth of Endothiapepsin Crystals for Serial Crystallography Experiments
Published on: February 4, 2021
An Energy Optimization Strategy Based on the Perfect Conformation of Prolyl Endopeptidase for Improving Catalytic
Xiaolin Li1, Yalong Cong1, Mingzhe Ma1
1Shanghai Engineering Research Center of Molecular Therapeutics & New Drug Development, Shanghai Key Laboratory of Green Chemistry & Chemical Process, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200062, China.
Abstract:
Prolyl endopeptidases (PEPs) hydrolyze proteins to yield bioactive peptides and are effective in the treatment of celiac disease. However, the catalytic efficiency of PEPs still has the potential to be improved, which could further strengthen their industrial and therapeutic applications. Herein, a novel rational design strategy based on a "near-attack conformation" of the catalytic state of PEP was adopted. Constrained dynamic simulations were applied, followed by the virtual screening of potentially favorable mutants according to their binding free energy. We redesigned Sphaerobacter thermophiles PEP with high-temperature activity/stability, a wide range of pH stabilities, and high proline specificity. As a result, the kcat value of two PEP mutants (I462W and Q560Y) increased by 208.2 and 150.1%, respectively, and the kcat/KM increased by 32.7 and 6.3%, respectively. These data revealed that the PEP mutants had improved catalytic efficiency and that our strategy can be applied for enzyme engineering.
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