title: Mapping of the thrombin des-ETW conformation by using site-directed mutants of hirudin. Evidence for the induction of nonlocal modifications by mutagenesis.
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)
authors.affiliation: Department of Haematology, University of Cambridge, England.
authors.forename: B F
authors.initials: BF
authors.lastname: Le Bonniec
authors.forename: A
authors.initials: A
authors.lastname: Betz
authors.forename: E R
authors.initials: ER
authors.lastname: Guinto
authors.forename: C T
authors.initials: CT
authors.lastname: Esmon
authors.forename: S R
authors.initials: SR
authors.lastname: Stone
mesh_terms: D000595:Amino Acid Sequence; D006629:Hirudins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism
chemical_list: D006629:Hirudins; D011994:Recombinant Proteins; D013917:Thrombin