Our laboratory has discovered the Nat-dependent allosteric enhancement of catalytic activity in thrombin and related serine proteases involved in blood coagulation and the complement system, identified the Na+ binding site and elucidated the importance of Na+ binding in the function and evolution of serine proteases. The proposed research project is aimed at gaining a detailed understanding of how Na+ is recognized by thrombin and how Na+ binding allosterically influences the structural determinants of activity and specificity of the enzyme. We will use a combination of kinetic and site-directed mutagenesis studies to dissect the structural determinants of the monovalent cation specificity of thrombin and to identify thrombin residues that are under the influence of Na+ binding and control recognition of physiologic substrates within the active site. Residues specifically involved in the binding and catalysis of fibrinogen, protein C and the thrombin receptors will be identified, thereby enabling a complete molecular dissection of thrombin multiple functions in the blood. We will also exploit the knowledge gained from the proposed research project on thrombin to rationally engineering Na+ binding and enhanced catalytic activity in the fibrinolytic enzyme tissue plasminogen activator. These studies will produce more proficient derivatives of the enzyme that may benefit the current treatment of acute myocardial infarction and stroke. Developments from the proposed research plan will broaden our understanding of the molecular aspects of thrombin function and regulation, will impact on the treatment and prevention of thrombotic disorders in which thrombin is directly involved, and will carry over to the study of other proteases in the blood coagulation cascade. By defining the rules for Na+ specificity in thrombin, these studies will generate important new knowledge relevant to allosteric proteins and enzymes activated by monovalent cations in general. Finally, these studies will demonstrate that proteases with enhanced catalytic activity can be engineered rationally to benefit areas of medical and biotechnological importance.
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