Gase A, Hartmann M, Gührs K H, Röcker A, Collen D, Behnke D, Schlott B
Institute of Molecular Biotechnology, Jena, Germany.
Thromb Haemost. 1996 Nov;76(5):755-60.
Structure/function relationships in the activation of plasminogen with staphylokinase were studied using mutants of recombinant staphylokinase (Sak42D). Deletion of up to 10 NH2-terminal amino acids (Sak42D delta N10) did not affect plasminogen activation, but removal of 11 amino acids completely abolished the ability to activate plasminogen. Elimination of potential plasmin cleavage sites in the NH2-terminal region yielding mutants Sak42D(K8H,K10H,K11H) and Sak42D(K6H,K8H,K11H) did not alter the rate of the exposure of a proteolytically active site (amidolytic activity) in equimolar mixtures with plasminogen, but destroyed the plasminogen activator properties of these muteins. Deleting two residues following the preferred processing site at position 10 (Sak42 delta (K11,G12)) resulted in a mutein also inactive in plasminogen activation. Removal of the COOH-terminal Lys136, yielding Sak42D delta C1, or of Lys135 and Lys136 in Sak42D delta C2 resulted in proteins with strongly reduced plasminogen activation capacity. In contrast, substitution of Lys135 and Lys136 with Ala in Sak42D(K135A,K136A) did not affect activation. Cyanogen bromide cleavage of Sak42D(M26L,E61M,D82E) produced a 61 amino acid NH2-terminal and a 65 amino acid COOH-terminal fragment which did not activate plasminogen, but bound to plasminogen with affinity constants Ka of 4.0 x 10(5) M-1 and 1.4 x 10(7) M-1, respectively (as compared to a Ka of 1.1 x 10(8) M-1 for Sak42D). These results indicate that Lys11 and the COOH-terminal region of staphylokinase play a key role in the activation of plasminogen.
利用重组葡萄球菌激酶（Sak42D）的突变体研究了葡萄球菌激酶激活纤溶酶原的结构/功能关系。删除多达10个氨基末端氨基酸（Sak42D delta N10）并不影响纤溶酶原的激活，但去除11个氨基酸则完全消除了激活纤溶酶原的能力。消除氨基末端区域潜在的纤溶酶切割位点，产生突变体Sak42D（K8H、K10H、K11H）和Sak42D（K6H、K8H、K11H），在与纤溶酶原的等摩尔混合物中，并未改变蛋白水解活性位点暴露的速率（酰胺水解活性），但破坏了这些突变蛋白的纤溶酶原激活剂特性。在第10位的首选加工位点之后删除两个残基（Sak42 delta（K11，G12）），产生的突变蛋白在纤溶酶原激活中也无活性。去除羧基末端的Lys136，产生Sak42D delta C1，或在Sak42D delta C2中去除Lys135和Lys136，得到的蛋白质纤溶酶原激活能力大幅降低。相比之下，在Sak42D（K135A，K136A）中将Lys135和Lys136替换为Ala并不影响激活。Sak42D（M26L，E61M，D82E）经溴化氰裂解产生一个61个氨基酸的氨基末端片段和一个65个氨基酸的羧基末端片段，它们不激活纤溶酶原，但分别以4.0×10⁵ M⁻¹和1.4×10⁷ M⁻¹的亲和常数Ka与纤溶酶原结合（相比之下，Sak42D的Ka为1.1×10⁸ M⁻¹）。这些结果表明，葡萄球菌激酶的Lys11和羧基末端区域在纤溶酶原激活中起关键作用。
