Functional roles of streptokinase C-terminal flexible peptide in active site formation and substrate recognition in plasminogen activation.
Functional roles of streptokinase C-terminal flexible peptide in active site formation and substrate recognition in plasminogen activation.
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链激酶 C 末端柔性肽在纤溶酶原激活中活性位点形成和底物识别中的功能作用。
DOI：
10.1021/bi026746m
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Zhang,XuejunC
中科院分区：
文献类型：
--
作者：
Zhai,Peng;Wakeham,Nancy;Loy,JeffreyA;Zhang,XuejunC
细菌蛋白链激酶 (SK) 将人纤溶酶原 (Pg) 激活为纤溶蛋白酶纤溶酶 (Pm)。 SK C 末端结构域的大约 40 个残基在与 Pm 催化结构域复合的 SK 晶体结构中是可移动的，并且该 C 尾部的功能仍然难以捉摸。为了更好地定义其在 Pg 激活中的作用，我们构建并表征了三个分别含有 SK 残基 1−378、1−386 和 1−401 的 C 端截短突变体。随着更多的 C 末端被删除，它们表现出逐渐降低的酰胺分解活性和 Pg 激活剂活性，以及对 Pg 的结合亲和力略有降低。与全长 SK 相比，最短的构建体 SK1-378 的酰胺分解活性 (kcat/KM) 降低 80%，Pg 激活剂活性降低 80%，Pg 催化结构域的解离常数增加 30%。 C端截短突变并没有减弱SK-Pm复合物对α2-抗纤溶酶的抵抗力。使用纯化的 C 尾肽来挽救截短突变体活性丧失的尝试失败了，这表明 SK C 端肽的完整性对于 SK 的完整功能非常重要。
The bacterial protein streptokinase (SK) activates human plasminogen (Pg) into the fibrinolytic protease plasmin (Pm). Roughly 40 residues from the SK C-terminal domain are mobile in the crystal structure of SK complexed with the catalytic domain of Pm, and the functions of this C-tail remain elusive. To better define its roles in Pg activation, we constructed and characterized three C-terminal truncation mutants containing SK residues 1−378, 1−386, and 1−401, respectively. They exhibit gradually reduced amidolytic activity and Pg-activator activity, as well as marginally decreased binding affinity toward Pg, as more of the C-terminus is deleted. As compared with full-length SK, the shortest construct, SK1-378, exhibits an 80% decrease in amidolytic activity (kcat/KM), an 80% decrease in Pg-activator activity, and a 30% increase in the dissociation constant toward the Pg catalytic domain. The C-terminal truncation mutations did not attenuate the resistance of the SK−Pm complex to α2-antiplasmin. Attempts at using a purified C-tail peptide to rescue the activity loss of the truncation mutants failed, suggesting that the integrity of the SK C-terminal peptide is important for the full function of SK.