Quoc D. Dang

The Na+ Binding Site of Thrombin

Thrombin is an allosteric serine protease existing in two forms, slow and fast, targeted toward anticoagulant and procoagulant activities. The slow → fast transition is induced by Na+ binding to a site contained within a cylindrical cavity formed by three antiparallel β-strands of the B-chain (Met-Tyr, Lys-Tyr, and Val-Gly) diagonally crossed by the Glu-Glu strand. The site is shaped further by the loop connecting the last two β-strands and is located more than 15 Å away from the catalytic triad. The cavity traverses through thrombin from the active site to the opposite surface and contains Asp of the primary specificity site near its midpoint. The bound Na+ is coordinated octahedrally by the carbonyl oxygen atoms of Tyr, Arg, and Lys, 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 (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.

Molecular mechanisms of thrombin function

Abstract. The discovery of thrombin as a Na+-dependent allosteric enzyme has revealed a novel strategy for regulating protease activity and specificity. The allosteric 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.

Selective Loss of Fibrinogen Clotting in a Loop-less Thrombin

The autolysis loop of thrombin comprises nine residues, from Glu146 to Lys149e, five of which (Ala149a–Lys149e) are inserted relative to trypsin and chymotrypsin. Deletion of the insertion Ala149a–Lys149e causes no significant change in the properties of the enzyme, except for a slight enhancement of protein C activation. Deletion of the entire Glu146–Lys149e loop, however, reduces fibrinogen clotting 240-fold, but decreases protein C activation only 2-fold. This loop-less mutant is de facto an exclusive activator of protein C, having lost the primary procoagulant function of thrombin. Because the autolysis loop affects fibrinogen binding, but not protein C activation, it provides a target for new drugs designed to suppress exclusively the procoagulant activity of thrombin.

Theory of allosteric effects in serine proteases.

The classical Botts-Morales theory for the action of a modifier on the catalytic properties of an enzyme has been extended to deal with allosteric effects in serine proteases. The exact analytical solution derived for the linkage scheme at steady state provides a rigorous framework for the study of many biologically relevant systems, including enzymes activated by monovalent cations and cofactor-controlled protease-zymogen interactions in blood coagulation. When the enzyme obeys Michaelis-Menten kinetics, the exact solution of the kinetic linkage scheme simplifies considerably. Of particular importance for practical applications is a simple equation expressing the dependence of the specificity constant of the enzyme, kcat/Km, on the concentration of the modifier, from which the equilibrium binding constant for the formation of the enzyme-modifier complex can be estimated. Analysis of the allosteric changes in thrombin activity induced by thrombomodulin and Na+ in terms of this equation yields accurate determinations of the equilibrium binding constants for both effectors.

A simple activity assay for thrombin and hirudin

Cloning of the thrombin cDNA has made it possible to study thrombin function by site-directed mutagenesis. Quantitative results from studies of thrombin mutants are often hindered by difficulties in assaying the enzyme activity. The high enzyme concentrations required for activity determination by standard methods limit their usefulness to thrombin mutants that cannot be readily produced in large quantities. We have developed a novel method using the synthetic substrate S-2238 and hirudin, a tight-binding inhibitor of thrombin, that allows for the active-site titration of thrombin at concentrations as low as 20 pM, with an error of ≤5%. In addition, hirudin activity can be determined by this method to concentrations as low as 40 pM, with an error of ≤5%.

[5] Linkage at steady state: Allosteric transitions of thrombin

Publisher Summary This chapter illustrates the importance of exploring linkage at steady state and the limitations of the equilibrium description. The exact analytical solution for the kinetic linkage scheme describing allosteric effects in serine proteases of the blood coagulation cascade and use this scheme to characterize energetically the allosteric properties of thrombin. The control of thrombin activity by allosteric effectors such as Na+ and the hirudin tail binding to the fibrinogen recognition site demonstrates that a great deal of information can be obtained from linkage studies under nonequilibrium conditions. In the case of thrombin, the linkage between important structural domains of the enzyme is dominated by the kinetic, rather than the equilibrium, components. The exact solution of the linkage scheme for serine proteases in the presence of an allosteric effector, as an extension of the Botts-Morales treatment of the action of a modifier is presented. The solution reveals the substantial complexity of linked functions at steady state and, at the same time, provides a convincing example of how macromolecules can exploit more complicated pathways of communication to accomplish biological function. The treatment sets the stage for a quantitative analysis of allosteric effects that dominate the blood coagulation cascade. It also provides the necessary framework for casting protein-protein interactions in this biologically relevant system.