MAPPING OF THE THROMBIN DES-ETW CONFORMATION BY USING SITE-DIRECTED MUTANTS OF HIRUDIN - EVIDENCE FOR THE INDUCTION OF NONLOCAL MODIFICATIONS BY MUTAGENESIS

Deletion of Glu146, Thr147, and Trp148 from thrombin dramatically alte rs its interactions with substrates, ligands, and inhibitors, and the changes appear to result from nonlocal modification of thrombin's stru cture [Le Bonniec, B. F., Guinto, E. R., & Esmon, C. T. (1992) J. Biol . Chem. 267, 19341-19348]. In an attempt to localize conformational ch ange in this thrombin mutant (des-ETW), its affinity for 43 site-direc ted mutants of hirudin have been compared with that of native thrombin . The inhibition constants (K(I)) of recombinant and natural hirudin w ith des-ETW were found to be almost-equal-to 2300-fold higher than tho se 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 test ed, the difference in binding energy between thrombin and des-ETW [DEL TADELTAG(b)degrees(IIa-ETW)] was about 19.9 kJ mol-1 and comparable to that of hirudin; in particular, all mutations in the C-terminal regio n of the inhibitor did not affect DELTADELTAG(b)degrees(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 DELTADELTAG(b)degrees(IIa-E TW) which were 2 to 10 kJ mol-1 lower than that for hirudin. Furthermo re, a Leu15'-->Ala hirudin mutant behaved, with des-ETW, as a partial competitive inhibitor rather than a tight-binding inhibitor, in contra st to all other hirudin variants tested. Results are interpreted in te rms of the thrombin-hirudin tertiary structure, and it is concluded th at the des-ETW mutation at least affects the conformations of (1) the catalytic serine, (2) the primary specificity pocket, and (3) the S2 b inding subsite. These nonlocal modifications of thrombin's structure s ubsequent to the des-ETW mutation may be caused by loss of the interna l salt bridge between Glu146 and Arg221A that seems to play a key role in thrombin's functions.