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  • Title: Mapping of the thrombin des-ETW conformation by using site-directed mutants of hirudin. Evidence for the induction of nonlocal modifications by mutagenesis.
    Author: Le Bonniec BF, Betz A, Guinto ER, Esmon CT, Stone SR.
    Journal: Biochemistry; 1994 Apr 05; 33(13):3959-66. PubMed ID: 8142400.
    Abstract:
    Deletion of Glu146, Thr147, and Trp148 from thrombin dramatically alters its interactions with substrates, ligands, and inhibitors, and the changes appear to result from nonlocal modification of thrombin's structure [Le Bonniec, B. F., Guinto, E. R., & Esmon, C. T. (1992) J. Biol. Chem. 267, 19341-19348]. In an attempt to localize conformational change in this thrombin mutant (des-ETW), its affinity for 43 site-directed mutants of hirudin have been compared with that of native thrombin. The inhibition constants (KI) of recombinant and natural hirudin with des-ETW were found to be approximately 2300-fold higher than those with thrombin. The reduced affinity was predominantly the result of an increase in the dissociation rate constant rather than a decrease in the association rate constant. For most of the hirudin mutants tested, the difference in binding energy between thrombin and des-ETW [delta delta Gbo(IIa-ETW)] was about 19.9 kJ mol-1 and comparable to that of hirudin; in particular, all mutations in the C-terminal region of the inhibitor did not affect delta delta Gbo(IIa-ETW). Thus, the conformation of thrombin's anion-binding exosite seems unaltered by the des-ETW mutation. In contrast, hirudin substitutions involving Val1', Val2', Thr4', Asp5', and Ser19', as well as the addition of an N-terminal glycine, resulted in values of delta delta Gbo(IIa-ETW) which were 2 to 10 kJ mol-1 lower than that for hirudin. Furthermore, a Leu15'-->Ala hirudin mutant behaved with des-ETW, as a partial competitive inhibitor rather than a tight-binding inhibitor, in contrast to all other hirudin variants tested.(ABSTRACT TRUNCATED AT 250 WORDS)
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