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

Site-directed mutagenesis has been used to construct a cDNA that encod es a recombinant variant hu an plasminogen (hPg) containing a Pro-611 --> Ile mutation (MrhPg). The mutein was expressed in recombinant bacu lo-virus-infected Spodoptera frugiperda cells (IPLB-SF-21AE), and puri fied. After activation of this zymogen to its corresponding form of th e serine protease plasmin (MrhPm), this latter enzyme was essentially inactive toward an amide plasmin substrate, most likely from alteratio n of the spatial relationships of the active-site His-603 to its partn ers of the catalytic triad, Asp-646 and Ser-741. Partial amidolytic ac tivity of MrhPm was restored as a consequence of imidazole addition to the assay medium, due to an increase in the catalytic constant k(cat) of the enzyme. The serine protease inhibitor, diisopropylphosphofluor idate, when preincubated with MrhPm, did not inhibit restoration of it s amidolytic activity with imidazole, whereas diisopropylphosphofluori date did inhibit the amidolytic activity of MrhPm in the presence of i midazole. This result implies that His-603 directly influences the nuc leophilic character of Ser-741. When imidazole was pretreated with alp ha-N-tosyl-L-lysine chloromethyl ketone, the ability of this imidazole solution to restore amidolytic activity to MrhPm was eliminated, sugg esting that N(alpha)-(p-tosyl)lysine chloromethyl ketone directs into the binding pocket a derivatized form of imidazole, which is ineffecti ve as an His-603 substitute. These results indicate that the conformat ional reorientation of His-603 results in a malfunctional catalytic tr iad in the serine protease MrhPm, thus leading to an inactive enzyme d espite the presence of all three essential amino acids of the catalyti c, triad. Addition of extramolecular imidazole restores a portion of t he amidolytic activity of this mutant enzyme. These data also argue fo r an enzyme mechanism in which the active-center His-603 residue direc tly influences the nucleophilicity of the active-site Ser 741 residue.