Deletion of Ile1 changes the mechanism of streptokinase: evidence for the molecular sexuality hypothesis.

Plasminogen (Plgn) is usually activated by proteolytic cleavage of Arg561-Val562. The new N-terminal amino group of Val562 forms a salt bridge with Asp740, creating the active protease plasmin (Pm). However, streptokinase (SK) binds to Plgn, generating an active protease in a poorly understood, nonproteolytic process. We hypothesized that the N-terminus of SK, Ile1, substitutes for the N-terminal Val562 of Pm, forming an analogous salt bridge with Asp740. SK initially forms an inactive complex with Plgn, which subsequently rearranges to create an active complex; this rearrangement is rate limiting at 4 degrees C. SK.Plgn efficiently hydrolyzes amide substrates at 4 degrees C, although DeltaIle1-SK. Plgn has no amidolytic activity. DeltaIle1-SK prevents formation of wild-type SK.Plgn. These results indicate that DeltaIle1-SK forms the initial inactive complex with plasminogen, but cannot form the active complex. However, when the experiment is performed at 37 degrees C, amidolytic activity is observed when DeltaIle1-SK is added to plasminogen. SDS-PAGE analysis demonstrates that the amidolytic activity results from the formation of DeltaIle1-SK.Pm. To further demonstrate that the activity of DeltaIle1-SK requires the conversion of Plgn to Pm, we characterized the reaction of SK with a mutant microplasminogen, Arg561Ala-microPlgn, that cannot be converted to microplasmin. Amidolytic activity is observed when Arg561Ala-microPlgn is incubated with wild-type SK at 37 degrees C; however, no amidolytic activity is observed in the presence of DeltaIle1-SK. These observations demonstrate that the amidolytic activity of DeltaIle1-SK at 37 degrees C requires the conversion of Plgn to Pm. Our findings indicate that Ile1 of SK is required for the nonproteolytic activation of Plgn by SK and are consistent with the hypothesis that Ile1 of SK substitutes for Val562 of Pm.