Mutant N143P reveals how Na+ activates thrombin

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• The precise molecular mechanism by which sodium ions (Na(+)) activate thrombin is not fully understood.
• Classical enzyme kinetics models require modification to incorporate the effects of Na(+) on thrombin's activity.

Purpose of the Study:

• To elucidate the allosteric mechanism of Na(+) activation in thrombin.
• To investigate the role of specific residues and structural changes in Na(+)-dependent thrombin activity.

Main Methods:

• Kinetic analysis using an extended Botts-Morales theory.
• Characterization of a thrombin mutant (N143P) with altered Na(+) sensitivity.
• X-ray crystallography to determine the structural basis of Na(+) binding and activation.

Main Results:

• The N143P mutant binds Na(+) but lacks Na(+)-dependent enhancement of catalytic activity (k(cat)).
• Crystal structures reveal that Pro(143) disrupts a critical hydrogen bond, affecting the Glu(192)-Gly(193) peptide bond orientation.
• Na(+) binding stabilizes the correct orientation of the Glu(192)-Gly(193) peptide bond, essential for oxyanion hole architecture.

Conclusions:

• Sodium ions activate thrombin by ensuring the proper orientation of the Glu(192)-Gly(193) peptide bond, likely through stabilizing interactions.
• This Na(+) activation mechanism, dependent on the 143-192 hydrogen bond and oxyanion hole integrity, is likely conserved in other trypsin-like proteases.