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首页> 外文期刊>Biochemistry >Deletion of Ile1 changes the mechanism of streptokinase: evidence for the molecular sexuality hypothesis.
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Deletion of Ile1 changes the mechanism of streptokinase: evidence for the molecular sexuality hypothesis.

机译：Ile1的删除改变了链激酶的机制：分子性假说的证据。

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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.
机译：纤溶酶原（Plgn）通常通过Arg561-Val562的蛋白水解裂解而激活。 Val562的新N末端氨基与Asp740形成盐桥，从而形成活性蛋白酶纤溶酶（Pm）。但是，链激酶（SK）与Plgn结合，在一个鲜为人知的非蛋白水解过程中产生了一种活性蛋白酶。我们假设SK的N末端Ile1替代了Pm的N末端Val562，与Asp740形成了类似的盐桥。 SK最初与Plgn形成非活动复合体，随后重新排列以创建活动复合体。这种重排限制了在4摄氏度下的速率。尽管DeltaIle1-SK，SK.Plgn在4摄氏度下有效地水解了酰胺底物。 Plgn没有酰胺分解活性。 DeltaIle1-SK可防止野生型SK.Plgn的形成。这些结果表明，DeltaIle1-SK与纤溶酶原形成了初始的非活性复合物，但不能形成活性复合物。但是，在37摄氏度下进行实验时，将DeltaIle1-SK添加到纤溶酶原中可观察到酰胺分解活性。 SDS-PAGE分析表明，酰胺水解活性是由DeltaIle1-SK.Pm的形成引起的。为了进一步证明DeltaIle1-SK的活性需要将Plgn转化为Pm，我们表征了SK与无法转化为微纤溶酶的突变型微纤溶酶原Arg561Ala-microPlgn的反应。当将Arg561Ala-microPlgn与野生型SK在37°C孵育时，可观察到酰胺分解活性。然而，在DeltaIle1-SK存在下没有观察到酰胺分解活性。这些观察表明，DeltaIle1-SK在37摄氏度的酰胺分解活性需要Plgn转化为Pm。我们的发现表明，SK的Ile1是SK进行Plgn的非蛋白水解激活所必需的，并且与SK的Ile1替代Pm的Val562的假设是一致的。

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