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

Plasminogen (Plgn) is usually activated by proteolytic cleavage of Arg561-V al562. The new N-terminal amino group of Val562 forms a salt bridge with As p740, creating the active protease plasmin (Pm). However, streptokinase (SK ) binds to Plgn, generating an active protease in a poorly understood, nonp roteolytic process. We hypothesized that the N-terminus of SK, Ile1, substi tutes for the N-terminal Val562 of Pm, forming an analogous salt bridge wit h Asp740. SK initially forms an inactive complex with Plgn, which subsequen tly rearranges to create an active complex; this rearrangement is rate limi ting at 4 degrees C. SK.Plgn efficiently hydrolyzes amide substrates at 4 d egrees C, although Delta Ile1-SK.Plgn has no amidolytic activity. Delta Ile 1-SK prevents formation of wild-type SK.Plgn. These results indicate that D elta Ile1-SK forms the initial inactive complex with plasminogen, but canno t form the active complex. However, when the experiment is performed at 37 degrees C, amidolytic activity is observed when Delta Ile1-SK is added to p lasminogen. SDS-PAGE analysis demonstrates that the amidolytic activity res ults from the formation of Delta Ile1-SK.Pm. To further demonstrate that th e activity of Delta Ile1-SK requires the conversion of Plgn to Pm, we chara cterized the reaction of SK with a mutant microplasminogen, Arg561Ala-mu Pl gn, that cannot be converted to microplasmin. Amidolytic activity is observ ed when Arg561Ala-mu Plgn is incubated with wild-type SK at 37 DC; however, no amidolytic activity is observed in the presence of Delta Ile1-SK. These observations demonstrate that the amidolytic activity of Delta Ile1-SK at 37 degrees C requires the conversion of Plgn to Pm. Our findings indicate t hat 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 Val5 62 of Pm.