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Mechanistic studies on thrombin catalysis.

S R Stone1, A Betz, J Hofsteenge

  • 1Friedrich Miescher-Institut, Basel, Switzerland.

Biochemistry
|October 15, 1991
PubMed
Summary
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Human alpha-thrombin

Area of Science:

  • Biochemistry
  • Enzymology
  • Pharmacology

Background:

  • Human alpha-thrombin is a key enzyme in blood coagulation.
  • Understanding its substrate interactions is crucial for developing anticoagulants.
  • The kinetic mechanism of substrate cleavage by alpha-thrombin has been studied.

Purpose of the Study:

  • To investigate the kinetic mechanism of human alpha-thrombin's cleavage of four p-nitroanilide (pNA) substrates.
  • To determine substrate "stickiness" and its effect on enzyme kinetics.
  • To elucidate the role of specific amino acid residues in catalysis.

Main Methods:

  • Steady-state kinetic techniques were employed.
  • Solvent isotope and viscosity effects were used to assess substrate stickiness.

Related Experiment Videos

  • pH profiles of kcat/Km were analyzed to determine pKa values.

    • Main Results:

    • Substrates exhibited varying degrees of "stickiness," from nonsticky (benzoyl-Arg-pNA) to very sticky (D-Phe-pipecolyl-Arg-pNA).
    • Bell-shaped pH profiles of kcat/Km were observed for all substrates.
    • pKa values around 7.5 and 9.1 were determined for most substrates, attributed to His57 and Ile16.

    Conclusions:

    • Substrate stickiness influences the kinetic mechanism of alpha-thrombin.
    • The catalytic activity of alpha-thrombin is modulated by pH, with specific residues playing a key role.
    • The observed pKa values provide insights into the active site environment and substrate binding.