Breaching the conformational integrity of the catalytic triad of the serine protease plasmin: localized disruption of a side chain of His-603 strongly inhibits the amidolytic activity of human plasmin.

摘要

Site-directed mutagenesis has been used to construct a cDNA that encodes a recombinant variant human plasminogen (hPg) containing a Pro-611-->Ile mutation (MrhPg). The mutein was expressed in recombinant baculovirus-infected Spodoptera frugiperda cells (IPLB-SF-21AE), and purified. After activation of this zymogen to its corresponding form of the serine protease plasmin (MrhPm), this latter enzyme was essentially inactive toward an amide plasmin substrate, most likely from alteration of the spatial relationships of the active-site His-603 to its partners of the catalytic triad, Asp-646 and Ser-741. Partial amidolytic activity of MrhPm was restored as a consequence of imidazole addition to the assay medium, due to an increase in the catalytic constant kcat of the enzyme. The serine protease inhibitor, diisopropylphosphofluoridate, when preincubated with MrhPm, did not inhibit restoration of its amidolytic activity with imidazole, whereas diisopropylphosphofluoridate did inhibit the amidolytic activity of MrhPm in the presence of imidazole. This result implies that His-603 directly influences the nucleophilic character of Ser-741. When imidazole as pretreated with alpha-N-tosyl-L-lysine chloromethyl ketone, the ability of this imidazole solution to restore amidolytic activity to MrhPm was eliminated, suggesting that N alpha-(p-tosyl)lysine chloromethyl ketone directs into the binding pocket a derivatized form of imidazole, which is ineffective as an His-603 substitute. These results indicate that the conformational reorientation of His-603 results in a malfunctional catalytic triad in the serine protease MrhPm, thus leading to an inactive enzyme despite the presence of all three essential amino acids of the catalytic triad. Addition of extramolecular imidazole restores a portion of the amidolytic activity of this mutant enzyme. These data also argue for an enzyme mechanism in which the active-center His-603 residue directly influences the nucleophilicity of the active-site Ser 741 residue.