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 Pro(143) abrogates the important H-bond between the backbone N atom of residue 143 and the carbonyl O atom of Glu(192), which in turn controls the orientation of the Glu(192)-Gly(193) 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.