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 fe wer peptide bonds in macromolecular substrates. To probe the nature of the specificity of thrombin, a mutant has been constructed in which t he Trp(148) loop of thrombin has been replaced with the same loop of b ovine trypsin. This mutant was expressed in Escherichia coli as prethr ombin-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 acti vated 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 tha t 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 thro mbin was also compared to trypsin and wild-type recombinant thrombin f or the ability to cleave small peptide substrates. The Michaelis const ants (K-m) were found to be between 5- and 10-fold higher for thrombin ((148)), relative to wild-type recombinant thrombin, although the cata lytic 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 hydrol ysis of -phenyalanyl-L-pipecolyl-L-arginine-p-nitroaniline (a thrombin substrate) than that of trypsin. For N-benzoyl-L-isoleucyL- L-glutamy lglycyl-L-arginine-p-nitroaniline (a trypsin substrate) and carbobenzo xyglycylprolyl-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 yie ld an enzyme with more trypsinlike specificity, the Trp(148) loop is i mportant in the substrate binding and specificity of thrombin (on the basis of K-m and K-i).