Thrombin is an allosteric serine protease existing in two forms, slow and fast, targeted toward anticoagulant and procoagulant activities. The slow 3 fast transition is induced by Na , and Lys 224 , and by three highly conserved water molecules in the D-Phe-Pro-Arg chloromethylketone thrombin. The sequence in the Na ؉ binding loop is highly conserved in thrombin from 11 different species and is homologous to that found in other serine proteases involved in blood coagulation. Mutation of two Asp residues flanking Arg 221a (D221A/D222K) almost abolishes the allosteric properties of thrombin and shows that the Na ؉ binding loop is also involved in direct recognition of protein C and antithrombin.

Residue 225 in serine proteases is typically Pro or Tyr and specifies an important and unanticipated functional aspect of this class of enzymes. Proteases with Y225, like thrombin, are involved in highly specialized functions like blood coagulation and complement that are exclusively found in vertebrates. In these proteases, the catalytic activity is enhanced allosterically by Na+ binding. Proteases with P225, like trypsin, are typically involved in digestive functions and are also found in organisms as primitive as eubacteria. These proteases have no requirement 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 0 atom of residue 224 makes a key contribution to the coordination shell of the bound Na+ in thrombin, but is oriented in a manner incompatible with Na+ binding in trypsin 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 proteases. To directly test this hypothesis, we have engineered the thrombin mutant Y225P. This mutant has lost the ability to bind Na+ and behaves like the allosteric slow (Na+-free) form. The Na+-induced allosteric regulation also bears on the molecular evolution of serine proteases. A strong correlation exists between residue 225 and the codon used for the active site S195. Proteases with P225 typically use a TCN codon for S195, whereas proteases with Y225 use an AGY codon. It is proposed that serine proteases evolved from two main lineages: (i) TCN/P225 with a trypsin-like ancestor and (ii) AGY/Y225 with a thrombin-like ancestor. We predict that the Na+-induced allosteric regulation of catalytic activity can be introduced in the TCN/P225 lineage using the P225Y replacement.Serine proteases participate in key physiological functions like digestion and blood coagulation, fibrinolysis, and complement (1-3). Proteases involved in digestive processes, like trypsin, have wide specificity and are also found in organisms as primitive as eubacteria. In contrast, proteases involved in the more specialized functions of blood coagulation, fibrinolysis, and complement have narrow specificity and are found exclusively in vertebrates (4-7). Among these more specialized enzymes, the blood clotting protease thrombin has been studied in great detail (8). Activity and specificity of this enzyme are controlled in an allosteric fashion by the binding of Na+ to a single site (9). The Na+-bound form (fast form) has drastically enhanced catalytic properties toward small chromogenic substrates and fibrinogen and is responsible for the procoagulant role of the enzyme, whereas the Na+-free form (slow form) cleaves and activates more specifically the anticoagulant protein C (10).The Na+-induced switch in specificity from protein C to fibrinogen discovered for throm...

The discovery of thrombin as a Na(+)-dependent allosteric enzyme has revealed a novel strategy for regulating protease activity and specificity. The alllosteric nature of this enzyme influences all its physiologically important interactions and rationalizes a large body of structural and functional information. For the first time, a coherent mechanistic framework is available for understanding how thrombin interacts with fibrinogen, thrombomodulin and protein C, and how Na+ binding influences the specificity sites of the enzyme. This information can be used for engineering thrombin mutants with selective specificity towards protein C and for the rational design of potent active site inhibitors. Thrombin also serves as a paradigm for allosteric proteases. Elucidation of the molecular basis of the Na(+)-dependent allosteric regulation of catalytic activity, based on the residue present at position 225, provides unprecedented insights into the function and evolution of serine proteases. This mechanism represents one of the simplest and most important structure-function correlations ever reported for enzymes in general. All vitamin K-dependent proteases and some complement factors are subject to the Na(+)-dependent regulation discovered for thrombin. Na+ is therefore a key factor in the activation of zymogens in the coagulation and complement systems.

The discovery of the Na(+)-dependent allosteric regulation in serine proteases makes it possible to control catalytic activity and specificity in this class of enzymes in a way never considered before. We demonstrate that rational site-directed mutagenesis of residues controlling Na+ binding can profoundly after the properties of a serine protease. By suppressing Na+ binding to thrombin, we shift the balance between procoagulant and anticoagulant activities of the enzyme. Those mutants, compared to wild-type, have reduced specificity toward fibrinogen, but enhanced or slightly reduced specificity toward protein C. Because this engineering strategy targets a fundamental regulatory mechanism, it is amenable of extension to other enzymes of biological and pharmacological importance.

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