Mutant N143P Reveals How Na+ Activates Thrombin

The molecular mechanism of thrombin activation by Na⁺ remains elusive. Its kinetic formulation requires extension of the classical Botts-Morales theory for the action of a modifier on an enzyme to correctly account for the contribution of the E*, E, and E:Na⁺ forms. The extended scheme establishes that analysis of k_cat unequivocally identifies allosteric transduction of Na⁺ binding into enhanced catalytic activity. The thrombin mutant N143P features no Na⁺-dependent enhancement of k_cat yet binds Na⁺ with an affinity comparable to that of wild type. Crystal structures of the mutant in the presence and absence of Na⁺ confirm that Pro¹⁴³ abrogates the important H-bond between the backbone N atom of residue 143 and the carbonyl O atom of Glu¹⁹², which in turn controls the orientation of the Glu¹⁹²-Gly¹⁹³ peptide bond and the correct architecture of the oxyanion hole. We conclude that Na⁺ activates thrombin by securing the correct orientation of the Glu¹⁹²-Gly¹⁹³ peptide bond, which is likely flipped in the absence of cation. Absolute conservation of the 143–192 H-bond in trypsin-like proteases and the importance of the oxyanion hole in protease function suggest that this mechanism of Na⁺ activation is present in all Na⁺-activated trypsin-like proteases.