Die Rolle der Insertionsschleife um Tryptophan 148 in der Aktivität von 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 Trp148 loop of thrombin has been replaced with the same loop of bovine trypsin. This mutant was expressed in Escherichia coli as prethrombin-2(148) using a T7 expression system previously described for wild-type prethrombin-2 [DiBella et al. (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 microM(-1)s(-1) for thrombin(148), which was approximately 20% of that of recombinant thrombin (25 +/- 2.0 microM(-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 trypsin-like specificity, the Trp(148) loop is important in the substrate binding and specificity of thrombin (on the basis of K(m) and K(i)).

Abstract aus PubMed (NCBI) für den zitierten Eintrag, in der Originalsprache wiedergegeben. Maßgeblich ist die Fassung der Originalpublikation.

Das Abstract wird nicht übersetzt, damit die eigene Wortwahl der Autoren erhalten bleibt.

Zur Untersuchung der Thrombin-Spezifität wurde die Trp148-Schleife durch jene von Rindertrypsin ersetzt und die Mutante als Präthrombin-2(148) in Escherichia coli exprimiert. Nach Rückfaltung und Aktivierung mit Echis-carinatus-Gift betrug kcat/Km für die Freisetzung von Fibrinopeptid A aus Fibrinogen 4,5 ± 0,5 µM⁻¹s⁻¹, etwa 20 % des rekombinanten Thrombins (25 ± 2,0 µM⁻¹s⁻¹). Thrombin(148) wurde durch Hirudin schlechter gehemmt (Ki 500 pM gegenüber 12 pM beim rekombinanten Thrombin); die Autoren schlussfolgern, dass die Trp148-Schleife für Substratbindung und Spezifität wichtig ist.

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