The linkage between adenosine nucleotide binding and amidase activity in human alpha-thrombin

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Human alpha-thrombin is a key enzyme in blood coagulation.
• Adenosine nucleotides (AMP, ADP, ATP) are known to interact with various proteins.
• The effect of adenosine nucleotides on thrombin's amidase activity is not fully elucidated.

Purpose of the Study:

• To investigate the influence of adenosine nucleotides (AMP, ADP, ATP) on human alpha-thrombin amidase activity.
• To develop a phenomenological model describing the linkage between nucleotide binding and thrombin activity.
• To determine the free energy changes associated with these interactions.

Main Methods:

• Enzyme kinetics assays measuring amidase activity of human alpha-thrombin.
• Experiments conducted in the presence of varying concentrations of AMP, ADP, and ATP.
• Global analysis of experimental data to resolve free energy changes.

Main Results:

• Low concentrations of adenosine nucleotides increased thrombin activity by up to 30%.
• High concentrations ( > 5 mM) of adenosine nucleotides inhibited thrombin activity by up to 20%.
• Inhibition was concentration-dependent and reduced by increasing substrate concentration.

Conclusions:

• Adenosine nucleotide binding to a regulatory site induces a conformational transition, activating thrombin.
• Inhibition at high nucleotide concentrations is attributed to competitive binding at the catalytic site.
• A phenomenological model successfully describes the observed linkage between nucleotide binding and thrombin amidase activity.