Subtle differences in active site structure between bovine and human thrombins: ESR and fluorescence studies

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Bovine and human alpha-thrombin share high primary structure homology.
• Existing X-ray structures lack resolution to differentiate conformational nuances.
• Understanding dynamic conformations is crucial for comparative analysis.

Purpose of the Study:

• To compare the dynamic conformations of bovine and human alpha-thrombins in solution.
• To investigate active site interactions using spin-labeled inhibitors and fluorophores.
• To identify subtle differences not discernible by X-ray crystallography.

Main Methods:

• Utilized active site-directed fluorosulfonylphenyl spin-labeled inhibitors and fluorophores.
• Probed regions within 10-15 Å of the catalytic serine residue.
• Analyzed immobilization of nitroxide moieties and spectral properties of fluorophores.

Main Results:

• Nitroxide moieties showed greater immobilization in bovine thrombin compared to human thrombin.
• Indole binding interactions were largely conserved, but subtle topographical differences were suggested.
• Active site-directed fluorophores exhibited distinct spectral shifts (lambda emmax, lambda exmax) between species.

Conclusions:

• Dynamic conformational differences exist between bovine and human alpha-thrombins.
• Apolar binding regions or steric factors likely contribute to observed immobilization differences.
• Solution-based spectroscopic methods provide insights into ligand binding and hydrophobic interactions inaccessible to X-ray crystallography alone.