Interaction of hirudin with the dysthrombins Quick I and II.

The interaction of hirudin with the dysfunctional enzymes thrombin Quick I and II has been investigated. Natural and recombinant hirudin caused nonlinear competitive inhibition of thrombin Quick I. The results were consistent with thrombin Quick I existing in two forms that have different affinities for hirudin. The affinities of these forms for natural hirudin were respectively 10(4)- and 10(6)-fold lower than that of alpha-thrombin. In contrast, truncated hirudin molecules lacking the C-terminal tail of the molecule caused linear inhibition of thrombin Quick I. These results indicate that different modes of interaction of the two forms of thrombin Quick I with the C-terminal tail of hirudin were the cause of the nonlinear inhibition. Comparison of the dissociation constants of thrombin Quick I with the truncated and full-length forms of hirudin suggested that the interactions that normally occur between the C-terminal tail of hirudin and thrombin were completely disrupted with the low-affinity form of thrombin Quick I. Thrombin Quick II displayed an affinity for natural hirudin that was 10(3)-fold lower than that observed with alpha-thrombin. In contrast, it bound a mutant hirudin with altered N-terminal amino acids only 16-fold less tightly. These results are discussed in terms of structural alterations in the active-site cleft in thrombin Quick II.