Streptokinase binds preferentially to the extended conformation of plasminogen through lysine binding site and catalytic domain interactions
Streptokinase binds preferentially to the extended conformation of plasminogen through lysine binding site and catalytic domain interactions
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DOI：
10.1021/bi000594i
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发表时间：
2000-11-14
期刊：
影响因子：
2.9
通讯作者：
Bock, PE
中科院分区：
文献类型：
--
作者：
Boxrud, PD;Bock, PE
链激酶(SK)与纤溶酶原(PG)结合，激活酶原构象，使其转化为纤溶酶原纤溶酶(Pm)。用Enililihrillm Hinrlino研究了SK与一系列催化位点标记的荧光PG和Pm类似物的相互作用，以确定赖氨酸结合部位的相互作用，PG的扩展构象和紧凑构象之间的相关变化，以及蛋白酶域的激活对结合SK，SK与荧光标记的[Glu]PG(L)和[Lys]PG(L)的亲和力的贡献，而标记的[Lys]PML的解离常数分别为624nM+/-112nM和38+/-5nM，而标记的[Lys]PML结合的解离常数为11+/-2 pM，57000倍6-氨基己酸饱和赖氨酸结合部位对SK与标记的[Glu]PGL的结合没有影响，但使SK与标记的[Lys]PGL和[Lys]PML的结合分别减弱31倍和20倍。在低氯离子浓度下，当[Glu]PG呈扩展构象而不占据赖氨酸结合部位时，观察到SK与标记的[Glu]PGL的亲和力增加了23倍，这可以通过表达新的赖氨酸结合部位相互作用来定量解释。结果支持这样的结论：由于赖氨酸结合部位的相互作用，标记的[Glu]PG转变为[Lys]PG衍生物的扩展构象，SK对荧光PG和Pm类似物的亲和力提高了13-16倍，SK对激活的蛋白酶结构域的亲和力进一步提高了3100-3500倍。结果表明，SK与[Glu]PG的结合在SK激活机制的早期事件中导致了[Glu]PG向扩展构象的转变。
Binding of streptokinase (SK) to plasminogen (Pg) activates the zymogen conformationally and initiates its conversion into the fibrinolytic proteinase, plasmin (Pm). Enililihrillm hinrlino studies of SK interactions with a homologous series of catalytic site-labeled fluorescent Pg and Pm analogues were performed to resolve the contributions of lysine binding site interactions, associated changes between extended and compact conformations of Pg, and activation of the proteinase domain to the affinity for SK, SK bound to fluorescein-labeled [Glu]Pg(l) and [Lys]Pg(l) with dissociation constants of 624 +/- 112 and 38 +/- 5 nM, respectively, whereas labeled [Lys]Pml bound with a 57000-fold tighter dissociation constant of 11 +/- 2 pM. Saturation of lysine binding sites with 6-aminohexanoic acid had no effect on SK binding to labeled [Glu]Pgl, but weakened binding to labeled [Lys]Pgl and [Lys]Pml 31- and 20-fold, respectively. At low Cl- concentrations, where [Glu]Pg assumes the extended conformation without occupation of lysine binding sites, a 23-fold increase in the affinity of SK for labeled [Glu]Pgl was observed, which was quantitatively accounted for by expression of new lysine binding site interactions. The results support the conclusion that the SK affinity for the fluorescent Pg and Pm analogues is enhanced 13-16-fold by conversion of labeled [Glu]Pg to the extended conformation of the [Lys]Pg derivative as a result of lysine binding site interactions, and is enhanced 3100-3500-fold further by the increased affinity of SK for the activated proteinase domain. The results imply that binding of SK to [Glu]Pg results in transition of [Glu]Pg to an extended conformation in an early event in the SK activation mechanism.