Wang S, Reed G L, Hedstrom L
Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02454, USA.
Biochemistry. 1999 Apr 20;38(16):5232-40. doi: 10.1021/bi981915h.
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.
纤溶酶原（Plgn）通常通过Arg561-Val562的蛋白水解切割而被激活。Val562新形成的N末端氨基与Asp740形成盐桥，从而产生活性蛋白酶纤溶酶（Pm）。然而，链激酶（SK）与Plgn结合，在一个尚未完全理解的非蛋白水解过程中产生一种活性蛋白酶。我们推测，SK的N末端Ile1替代了Pm的N末端Val562，与Asp740形成类似的盐桥。SK最初与Plgn形成无活性复合物，随后重新排列形成活性复合物；这种重排在4℃时是限速步骤。SK.Plgn在4℃时能有效水解酰胺底物，尽管缺失Ile1的SK（DeltaIle1-SK）.Plgn没有酰胺水解活性。DeltaIle1-SK可阻止野生型SK.Plgn的形成。这些结果表明，DeltaIle1-SK与纤溶酶原形成初始无活性复合物，但不能形成活性复合物。然而，当在37℃进行实验时，将DeltaIle1-SK加入纤溶酶原中可观察到酰胺水解活性。SDS-PAGE分析表明，酰胺水解活性是由DeltaIle1-SK.Pm的形成所致。为进一步证明DeltaIle1-SK的活性需要Plgn转化为Pm，我们对SK与一种不能转化为微纤溶酶的突变型微纤溶酶原Arg561Ala-微Plgn的反应进行了表征。当Arg561Ala-微Plgn在37℃与野生型SK孵育时可观察到酰胺水解活性；然而，在DeltaIle1-SK存在的情况下未观察到酰胺水解活性。这些观察结果表明，DeltaIle1-SK在37℃时酰胺水解活性需要Plgn转化为Pm。我们的研究结果表明，SK的Ile1是SK对Plgn进行非蛋白水解激活所必需的，这与SK的Ile1替代Pm的Val562这一假设一致。
