Conformational differences between high clotting human alpha-thrombin and nonclotting gamma-thrombin

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Human alpha-thrombin is a key enzyme in blood coagulation.
• Gamma-thrombin is a proteolytically modified form of alpha-thrombin.
• Understanding conformational differences is crucial for enzyme function studies.

Purpose of the Study:

• To compare the conformations of human alpha-thrombin and gamma-thrombin.
• To elucidate the structural changes accompanying the conversion of alpha-thrombin to gamma-thrombin.
• To investigate the impact of these changes on enzyme compactness and catalytic site.

Main Methods:

• Circular dichroism (CD) spectroscopy to assess secondary structure.
• Solvent perturbation difference spectroscopy to quantify exposed aromatic residues (tryptophan and tyrosine).
• Chemical modification using dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide to assess residue reactivity.
• Inhibition studies with phenyl-methanesulfonyl fluoride to probe catalytic site conformation.

Main Results:

• Circular dichroism revealed a significant decrease in alpha-helical content during conversion from alpha-thrombin (5-7%) to gamma-thrombin (0-1%).
• Solvent perturbation indicated increased exposure of tryptophan and tyrosine residues in gamma-thrombin compared to alpha-thrombin.
• Gamma-thrombin showed greater reactivity of tryptophan residues and subtle differences in inhibitor blocking, suggesting a less compact overall structure and a tighter catalytic site.

Conclusions:

• The conversion of alpha-thrombin to gamma-thrombin involves substantial conformational alterations.
• Gamma-thrombin exhibits a less compact molecular structure than alpha-thrombin.
• These conformational changes, particularly in the catalytic site, may influence gamma-thrombin's enzymatic activity and substrate interactions.