El papel del bucle de inserción alrededor del triptófano 148 en la actividad de la trombina

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)).

Resumen tomado de PubMed (NCBI) para el registro citado, en el idioma original. La versión de referencia es la de la publicación original.

El resumen se deja sin traducir para conservar la redacción original de los autores.

Se sustituyó el bucle Trp148 de la trombina por el de la tripsina bovina y el mutante se expresó en Escherichia coli. Su kcat/Km para la liberación de fibrinopéptido A fue de 4,5 ± 0,5 µM⁻¹s⁻¹, cerca del 20 % del valor de la trombina recombinante (25 ± 2,0 µM⁻¹s⁻¹), y la hirudina lo inhibió menos (Ki 500 pM frente a 12 pM). Según los autores, el bucle Trp148 es importante para la unión al sustrato y la especificidad.

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