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The Na+ binding site of thrombin
E Di Cera1, E R Guinto, A Vindigni
1Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
The Journal of Biological Chemistry
|September 22, 1995
Summary
Sodium ion (Na+) binding induces a conformational change in thrombin, an allosteric serine protease. This transition, crucial for regulating anticoagulant and procoagulant activities, is mediated by a specific binding site within the enzyme.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Thrombin, a serine protease, exhibits allosteric behavior, existing in slow (anticoagulant) and fast (procoagulant) forms.
- The transition between these forms is modulated by sodium ion (Na+) binding.
Purpose of the Study:
- To elucidate the structural basis of Na+ binding and its role in thrombin's allosteric regulation.
- To investigate the functional implications of the Na+ binding site in thrombin's interaction with inhibitors.
Main Methods:
- Structural analysis of thrombin, focusing on the Na+ binding site within a beta-strand cavity.
- Site-directed mutagenesis (D221A/D222K) to probe the function of the Na+ binding loop.
Main Results:
- Identified a specific Na+ binding site within a cavity formed by antiparallel beta-strands, distant from the catalytic triad.
- Demonstrated that Na+ coordination involves specific amino acid residues (Tyr184a, Arg221a, Lys224) and water molecules.
- Showed that mutations in the Na+ binding loop significantly impair thrombin's allosteric properties and interaction with protein C and antithrombin.
Conclusions:
- Sodium ion binding is a key allosteric regulator of thrombin's enzymatic activity.
- The Na+ binding site and loop are critical for thrombin's conformational transitions and interactions with key regulatory proteins.