Schlott B, Gührs K H, Hartmann M, Röcker A, Collen D
Institute for Molecular Biotechnology, 07745 Jena, Germany.
J Biol Chem. 1997 Feb 28;272(9):6067-72. doi: 10.1074/jbc.272.9.6067.
Staphylokinase (Sak), a single-chain protein comprising 136 amino acids with NH2-terminal sequence,SSSFDKGKYKKGDDA forms a complex with plasmin, that is endowed with plasminogen activating properties. Plasmin is presumed to process mature (high molecular weight, HMW) Sak to low molecular weight derivatives (LMW-Sak), primarily by hydrolyzing the Lys10-Lys11 peptide bond, but the kinetics of plasminogen activation by HMW-Sak and LMW-Sak are very similar. Here, the requirement of NH2-terminal proteolysis of Sak for the induction of plasminogen activating potential was studied by mutagenesis of Lys10 and Lys11 in combination with NH2-terminal microsequence analysis of equimolar mixtures of Sak and plasminogen and determination of kinetic parameters of plasminogen activation by catalytic amounts of Sak. Substitution of Lys10 with Arg did not affect processing of the Arg10-Lys11 site nor plasminogen activation, whereas substitution with His resulted in cleavage of the Lys11-Gly12 peptide bond and abolished plasminogen activation. Substitution of Lys11 with Arg did not affect Lys10-Arg11 processing or plasminogen activation, whereas replacement with His did not prevent Lys10-His11 hydrolysis but abolished plasminogen activation. Substitution of Lys11 with Cys yielded an inactive processed derivative which was fully activated by aminoethylation. Deletion of the 10 NH2-terminal amino acids did not affect plasminogen activation, but additional deletion of Lys11 eliminated plasminogen activation. Thus generation of plasminogen activator potential in Sak proceeds via plasmin-mediated removal of the 10 NH2-terminal amino acids with exposure of Lys11 as the new NH2 terminus. This provides a structural basis for the hypothesis, derived from kinetic measurements, that plasminogen activation by Sak needs to be primed by plasmin and a mechanism for the high fibrin selectivity of Sak in a plasma milieu.
葡萄球菌激酶（Sak）是一种由136个氨基酸组成的单链蛋白，其氨基末端序列为SSSFDKGKYKKGDDA，它与纤溶酶形成复合物，具有纤溶酶原激活特性。据推测，纤溶酶主要通过水解Lys10-Lys11肽键将成熟的（高分子量，HMW）Sak加工成低分子量衍生物（LMW-Sak），但HMW-Sak和LMW-Sak激活纤溶酶原的动力学非常相似。在此，通过对Lys10和Lys11进行诱变，结合对Sak和纤溶酶原等摩尔混合物的氨基末端微序列分析以及测定催化量的Sak激活纤溶酶原的动力学参数，研究了Sak氨基末端蛋白水解对纤溶酶原激活潜能诱导的需求。用Arg取代Lys10既不影响Arg10-Lys11位点的加工，也不影响纤溶酶原激活，而用His取代则导致Lys11-Gly12肽键的裂解并消除了纤溶酶原激活。用Arg取代Lys11不影响Lys10-Arg11的加工或纤溶酶原激活，而用His取代虽不阻止Lys10-His11的水解，但消除了纤溶酶原激活。用Cys取代Lys11产生了一种无活性的加工衍生物，通过氨乙基化可使其完全激活。删除10个氨基末端氨基酸不影响纤溶酶原激活，但额外删除Lys11则消除了纤溶酶原激活。因此，Sak中纤溶酶原激活潜能的产生是通过纤溶酶介导去除10个氨基末端氨基酸，使Lys11暴露为新的氨基末端。这为基于动力学测量得出的假说提供了结构基础，即Sak激活纤溶酶原需要纤溶酶引发，也为Sak在血浆环境中对纤维蛋白具有高选择性的机制提供了依据。
