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Molecular dissection of Na+ binding to thrombin
Agustin O Pineda1, Christopher J Carrell, Leslie A Bush
1Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
The Journal of Biological Chemistry
|May 21, 2004
Summary
Sodium (Na+) binding to thrombin allosterically enhances its procoagulant functions. Key residues and a water network mediate this activation, revealing a new paradigm for enzyme regulation in blood coagulation.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Sodium (Na+) ions are known to influence the activity of thrombin, a key enzyme in blood coagulation.
- This influence is thought to be allosteric, involving communication between Na+ binding sites and substrate recognition regions.
Purpose of the Study:
- To identify the specific residues forming the allosteric core responsible for Na+ binding effects in thrombin.
- To elucidate the structural and mechanistic basis of Na+-mediated thrombin activation.
Main Methods:
- Alanine mutagenesis of 78 residues in thrombin.
- X-ray crystallography of Na+-free and Na+-bound thrombin, with and without an active site inhibitor.
- Analysis of allosteric communication pathways and water networks.
Main Results:
- Identified Asp-189, Glu-217, Asp-222, and Tyr-225 as the allosteric core linked to Na+ binding.
- Discovered that Asp-189 and Asp-221 transduce Na+ binding into enhanced catalytic activity.
- Observed conformational changes upon Na+ binding, including the formation of the Arg-187:Asp-222 ion pair and a rearranged water network connecting Na+ to Ser-195.
Conclusions:
- Na+ binding to thrombin induces conformational changes via an allosteric core and a critical water network, enhancing catalytic activity and procoagulant functions.
- This study establishes a new paradigm for allosteric regulation of thrombin and other Na+-activated enzymes in coagulation and immunity.