Deletion of Ile1 changes the mechanism of streptokinase: evidence for the molecular sexuality hypothesis.
Deletion of Ile1 changes the mechanism of streptokinase: evidence for the molecular sexuality hypothesis.
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Ile1 的缺失改变了链激酶的机制：分子性假设的证据。
DOI：
10.1021/bi981915h
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发表时间：
1999
期刊：
影响因子：
2.9
通讯作者：
Hedstrom,L
中科院分区：
文献类型：
--
作者：
Wang,S;Reed,GL;Hedstrom,L
纤溶酶原（Plgn）通常由Arg561−Val562的蛋白水解裂解激活。Val562新的n端氨基与Asp740形成盐桥，产生活性蛋白酶plasmin （Pm）。然而，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一种活性蛋白酶。我们假设SK的n端Ile1取代Pm的n端Val562，与Asp740形成类似的盐桥。SK最初与Plgn形成非活性复合体，Plgn随后重新排列形成活性复合体；这种重排在4°C时速率受限。SK·Plgn在4℃时能有效水解酰胺底物，但ΔIle1−SK·Plgn无酰胺水解活性。ΔIle1−SK阻止野生型SK·Plgn的形成。这些结果表明ΔIle1−SK与纤溶酶原形成初始的无活性复合物，但不能形成活性复合物。然而，当实验在37°C下进行时，当纤溶酶原中加入ΔIle1−SK时，观察到酶解活性。SDS - PAGE分析表明，酶解活性来源于ΔIle1−SK·Pm的形成。为了进一步证明ΔIle1−SK的活性需要Plgn转化为Pm，我们表征了SK与突变型微纤溶酶原Arg561Ala−μPlgn的反应，该突变型微纤溶酶原不能转化为微纤溶酶。Arg561Ala−μPlgn与野生型SK在37℃下孵育，观察到其酶解活性；然而，在ΔIle1−SK的存在下，没有观察到酶解活性。这些观察结果表明，ΔIle1−SK在37℃时的酶解活性需要将Plgn转化为Pm。我们的研究结果表明SK的Ile1是SK对Plgn的非蛋白水解激活所必需的，这与SK的Ile1替代Pm的Val562的假设是一致的。
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 °C. SK·Plgn efficiently hydrolyzes amide substrates at 4 °C, although ΔIle1−SK·Plgn has no amidolytic activity. ΔIle1−SK prevents formation of wild-type SK·Plgn. These results indicate that ΔIle1−SK forms the initial inactive complex with plasminogen, but cannot form the active complex. However, when the experiment is performed at 37 °C, amidolytic activity is observed when ΔIle1−SK is added to plasminogen. SDS−PAGE analysis demonstrates that the amidolytic activity results from the formation of ΔIle1−SK·Pm. To further demonstrate that the activity of ΔIle1−SK requires the conversion of Plgn to Pm, we characterized the reaction of SK with a mutant microplasminogen, Arg561Ala−μPlgn, that cannot be converted to microplasmin. Amidolytic activity is observed when Arg561Ala−μPlgn is incubated with wild-type SK at 37 °C; however, no amidolytic activity is observed in the presence of ΔIle1−SK. These observations demonstrate that the amidolytic activity of ΔIle1−SK at 37 °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.