The role of the insertion loop around tryptophan 148 in the activity of thrombin

Thrombin has trypsin-like specificity for Arg-Xaa and Lys-Xaa peptide bonds; however, it is much more specific than trypsin, cleaving far fewer peptide bonds in macromolecular substrates. To probe the nature of the specificity of thrombin, a mutant has been constructed in which the Trp(148) loop of thrombin has been replaced with the same loop of bovine trypsin. This mutant was expressed in Escherichia coli as prethrombin-21((148)) using a T7 expression system previously described for wild-type prethrombin-2 [DiBella et nl, (1995) J. Biol. Chem. 270, 163-169]. After refolding and purification, prethrombin-2((148)) was activated to thrombin((148)), with Echis carinatus snake venom, The k(cat)/K-m for the release of fibrinopeptide A from fibrinogen was 4.5 +/- 0.5 mu M(-1) s(-1) for thrombin((148)), which was similar to 20% of that of recombinant thrombin (25 +/- 2.0 mu M(-1) s(-1)). Thrombin ((148)), was inhibited less well by hirudin with a K-i of 500 pM compared to a value of 12 pM determined for recombinant thrombin. The mutant thrombin was also compared to trypsin and wild-type recombinant thrombin for the ability to cleave small peptide substrates. The Michaelis constants (K-m) were found to be between 5- and 10-fold higher for thrombin((148)), relative to wild-type recombinant thrombin, although the catalytic constants (k(cat)) for thrombin((148)) and recombinant thrombin remained relatively unchanged for all three substrates. Thrombin((148)) had a specificity constant (k(cat)/K-m) 2-fold higher for the hydrolysis of H-D-phenyalanyl-L-pipecolyl-L-arginine-p-nitroaniline (a thrombin substrate) than that of trypsin. For N-benzoyl-L-isoleucyL- L-glutamylglycyl-L-arginine-p-nitroaniline (a trypsin substrate) and N-carbobenzoxyglycylprolyl-L-arginine-p-nitroaniline (a substrate for both enzymes), the specificity constants for trypsin were 1000- and 16-fold higher, respectively, Although replacement of the Trp(148) loop does not yield an enzyme with more trypsinlike specificity, the Trp(148) loop is important in the substrate binding and specificity of thrombin (on the basis of K-m and K-i).