The gamma-loop of thrombin is a flexible, surface-accessible loop in free thrombin that appears to be one of several sites participating in the interaction of the enzyme with macromolecular substrates and inhibitors. Using limited proteolysis and intrinsic fluorescence measurements, we have studied changes in thrombin structure induced by small, site-specific ligands. Binding of a C-terminal peptide of hirudin to the anion-binding exosite of thrombin induced a structural change in the gamma-loop, which caused a 6-fold reduction in the susceptibility of the enzyme to limited proteolysis by elastase and chymotrypsin. Binding of several active site-specific thrombin inhibitors conferred an even greater protection from proteolysis at the gamma-loop. For example, the covalent complex of thrombin with D-Phe-Pro-Arg-CH2Cl was 95-fold less susceptible to cleavage by chymotrypsin than the free enzyme. Furthermore, binding of either exosite or active-site probes induced a common intrinsic fluorescence change in thrombin (a fractional increase of 0.13). These results are surprising because crystallographic studies indicate that direct contact between the bound probes and relevant residues of the gamma-loop is very unlikely. Thus we have identified an allosteric interaction that couples the active site of thrombin to the gamma-loop. An interaction of this nature may be one way in which thrombomodulin modulates the reactivity of thrombin.

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