Zymogen activation in the streptokinase–plasminogen complex

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 (mPlgn) 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 $ 10 4 -fold (Ile13Ala, 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 mPlgn 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.

Structure-Function Analysis of the Streptokinase Amino Terminus (Residues 1–59)

“…Incubation of peptides containing amino acids 1-59 of SK with inactive deletion mutants lacking these residues restores PA activity (8,9,13). Deletion of Ile 1 of SK markedly inhibits its capacity to induce an active site in plasminogen, which supports the hypothesis that establishment of a salt bridge between Ile 1 of SK and Asp 740 of plasminogen is necessary for SK to induce an active site in plasminogen by a nonproteolytic mechanism (11,14). Residues 1-59 also appear to play an important role in determining the capacity of SK to efficiently activate plasminogen in the absence of fibrin, a property that distinguishes SK from tissue-type plasminogen activator.…”

“…The experiment was performed three times with similar results. mation by a nonproteolytic mechanism (14). However, complexes of plasmin and SKs harboring mutations between positions 18 -24 were severely defective in PA activity, whereas complexes of SK I1A or SK-(2-414) and plasmin exhibited normal PA activity.…”

“…First, what residues play key roles in mediating the functional effects of the amino-terminal fragment on plasminogen activation? Although the role of Ile 1 of SK in plasminogen activation has been examined in some detail (11,14), less is known about the function of other specific residues (16 -19). Second, what is the mechanism by which complexes composed of plasminogen and SK mutants lacking portions of the amino terminus attain normal or nearly normal amidolytic activity, but only after a prolonged lag phase?…”

Resolution of Conformational Activation in the Kinetic Mechanism of Plasminogen Activation by Streptokinase

“…Studies demonstrating a critical role of the N terminus of SK in conformational activation support the "molecular sexuality" mechanism (34,56,57). In this mechanism, insertion of the SK N terminus into the N-terminal binding cleft in the Pg catalytic domain and salt bridge formation with Asp 194 (chymotrypsin numbering) triggers the activating conformational change.…”

The Mechanism of a Bacterial Plasminogen Activator Intermediate between Streptokinase and Staphylokinase

“…Although SUPA could not generate an active site in h-Pg, the b-plasmin⅐SUPA complex (12). Mutagenesis studies aimed at identifying the counterion have indicated that Ile 1 is required for nonproteolytic activation of Pg, while Lys 698 appears to be required for normal binding interactions during the formation of the initial SK⅐Pg complex (12,31). The conservation of the NH 2 -terminal motif (Ile-Xxx-Gly) between SUPA and all other reported SKs underlines the importance of this residue (12).…”