Pozzi N, Chen Z, Zapata F, Niu W, Barranco-Medina S, Pelc LA, Di Cera E. J Biol Chem. 2013 Apr 19;288(16):11601-10. Abstract: Trypsin-like proteases are synthesized as inactive zymogens and convert to the mature form upon activation by specific enzymes, often assisted by cofactors. Central to this paradigm is that the zymogen does not convert spontaneously to the mature enzyme, which in turn does not feed back to activate its zymogen form. In the blood, the zymogens prothrombin and prethrombin-2 require the prothrombinase complex to be converted to the mature protease thrombin, which is unable to activate prothrombin or prethrombin-2. Here, we show that replacement of key residues within the activation domain causes these zymogens to spontaneously convert to thrombin. The conversion is started by the zymogen itself, which is capable of binding ligands at the active site, and is abrogated by inactivation of the catalytic residue Ser-195. The product of autoactivation is functionally and structurally equivalent to wild-type thrombin. Zymogen autoactivation is explained by conformational selection, a basic property of the trypsin fold uncovered by structural and rapid kinetics studies. Both the zymogen and protease undergo a pre-existing equilibrium between active and inactive forms. The equilibrium regulates catalytic activity in the protease and has the potential to unleash activity in the zymogen to produce autoactivation. A new strategy emerges for the facile production of enzymes through zymogen autoactivation that is broadly applicable to trypsin-like proteases of biotechnological and clinical interest.