RESIDUE-225 DETERMINES THE NA-INDUCED ALLOSTERIC REGULATION OF CATALYTIC ACTIVITY IN SERINE PROTEASES()

Residue 225 in serine proteases is typically Pro or Tyr and specifies an important and unanticipated functional aspect of this class of enzy mes, Proteases with Y225, like thrombin, are involved in highly specia lized functions like blood coagulation and complement that are exclusi vely found in vertebrates, In these proteases, the catalytic activity is enhanced allosterically by Na+ binding, Proteases with P225, like t rypsin, are typically involved in digestive functions and are also fou nd in organisms as primitive as eubacteria. These proteases have no re quirement for Na+ or other monovalent cations. The molecular origin of this physiologically important difference is remarkably simple and is revealed by a comparison of the Na+ binding loop of thrombin with the homologous region of trypsin. The carbonyl O atom of residue 224 make s a key contribution to the coordination shell of the bound Na+ in thr ombin, but is oriented in a manner incompatible with Na+ binding in tr ypsin because of constraints imposed by P225 on the protein backbone. Pro at position 225 is therefore incompatible with Na+ binding and is a direct predictor of the lack of allosteric regulation in serine prot eases. To directly test this hypothesis, we have engineered the thromb in mutant Y225P. This mutant has lost the ability to bind Na+ and beha ves like the allosteric slow (Na+-free) form. The Na+-induced alloster ic regulation also bears on the molecular evolution of serine protease s. A strong correlation exists between residue 225 and the codon used for the active site S195, Proteases with P225 typically use a TCN codo n for S195, whereas proteases with Y225 use an AGY codon, It is propos ed that serine proteases evolved from two main lineages: (i) TCN/P225 with a trypsin-like ancestor and (ii) AGY/Y225 with a thrombin-like an cestor. We predict that the Na+-induced allosteric regulation of catal ytic activity can be introduced in the TCN/P225 lineage using the P225 Y replacement.