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Loop Electrostatics Asymmetry Modulates the Preexisting Conformational Equilibrium in Thrombin.

Nicola Pozzi1, Mirco Zerbetto, Laura Acquasaliente

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Altering thrombin

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Area of Science:

  • Biochemistry
  • Enzymology
  • Structural Biology

Background:

  • Thrombin (E) exists in equilibrium with an inactive form (E*), differing in active site accessibility.
  • Understanding the factors controlling this equilibrium is crucial for enzyme regulation.

Purpose of the Study:

  • To investigate how perturbing electrostatic properties affects the thrombin E*-E equilibrium.
  • To elucidate the structural consequences of altering key charged residues.

Main Methods:

  • Site-directed mutagenesis of Asp102 and Asp189 to Alanine (D102N, D189A).
  • X-ray crystallography to determine high-resolution structures of mutants.
  • Solution studies to analyze conformational changes.

Main Results:

  • Removal of negative charges at Asp102 or Asp189 shifts the equilibrium towards the inactive E* form.
  • Mutations cause a conformational shift in the 215-217 segment, hindering substrate access.
  • Structural analysis confirms stabilization of the E* form in D102N and D189A mutants.

Conclusions:

  • Perturbing electrostatic properties is a novel strategy to control the thrombin E*-E equilibrium.
  • This work establishes a new paradigm for regulating enzyme activity within the trypsin fold.
  • Findings have implications for understanding enzyme dynamics and designing inhibitors.