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Chemical compensation in macromolecular bridge-binding to thrombin
K P Hopfner1, Y Ayala, Z Szewczuk
1Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
Biochemistry
|March 30, 1993
Summary
Synthetic peptides were designed to inhibit thrombin by targeting both its catalytic pocket and fibrinogen-recognition site. A 13-carbon linker yielded nanomolar inhibition, revealing key binding energetics.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Thrombin is a key enzyme in blood coagulation.
- Developing effective thrombin inhibitors is crucial for treating thrombotic disorders.
- Synthetic peptides offer a modular approach to designing enzyme inhibitors.
Purpose of the Study:
- To investigate the binding energetics of novel bifunctional synthetic peptides targeting thrombin.
- To determine the optimal linker characteristics for potent thrombin inhibition.
- To elucidate the thermodynamic basis of inhibitor-enzyme interactions.
Main Methods:
- Steady-state enzyme kinetics measurements.
- Clotting assays to assess anticoagulant activity.
- Thermodynamic analysis (temperature dependence) of binding interactions.
Main Results:
- Eight synthetic peptides were synthesized and characterized as bifunctional thrombin inhibitors.
- A linker of 13 carbon atoms resulted in nanomolar inhibition (KI) against thrombin.
- Enthalpic and entropic contributions to inhibitor binding were quantified.
- Chemical compensation was observed for peptides binding to both enzyme sites.
- Hirudin binding to thrombin is an entropy-driven process, showing preferential binding over fibrinogen.
Conclusions:
- Bifunctional synthetic peptides can effectively inhibit thrombin with high potency.
- Linker length is critical for optimizing the inhibitory activity of these peptides.
- Thermodynamic analysis provides insights into the molecular mechanisms of thrombin inhibition.