Abstract
Plasminogen (Plgn) is usually activated by proteolysis of the Arg561–Val562 bond. The amino group of Val562 forms a salt‐bridge with Asp740, which triggers a conformational change producing the active protease plasmin (Pm). In contrast, streptokinase (SK) binds to Plgn to produce an initial inactive complex (SK·Plgn) which subsequently rearranges to an active complex (SK·Plgn*) although the Arg561–Val562 bond remains intact. Therefore another residue must substitute for the amino group of Val562 and provide a counterion for Asp740 in this active complex. Two candidates for this counterion have been suggested: Ile1 of streptokinase and Lys698 of Plgn. We have investigated the reaction of SK mutants and variants of the protease domain of microplasminogen (µPlgn) in order to determine if either of these residues is the counterion. The mutation of Ile1 of SK decreases the activity of SK·Plgn* by 100‐fold (Ile1Val) to ≥ 104‐fold (Ile1→Ala, Gly, Trp or Lys). None of these mutations perturb the binding affinity of SK, which suggests that Ile1 is not required for formation of SK·Plgn but is necessary for SK·Plgn*. The substitution of Lys698 of µPlgn decreases the activity of SK·Plgn* by only 10–60‐fold. In contrast with the Ile1 substitutions, the Lys698 mutations also decreased the dissociation constant of the SK complex by 15–50‐fold. These observations suggest that Lys698 is involved in formation of the initial SK·Plgn complex. These results support the hypothesis that Ile1 provides the counterion for Asp740.