Wang Haiyao, Yu Anna, Wiman Björn, Pap Sarolta
Department of Clinical Chemistry and Blood Coagulation, Karolinska Hospital, Karolinska Institute, Stockholm, Sweden.
Eur J Biochem. 2003 May;270(9):2023-9. doi: 10.1046/j.1432-1033.2003.03578.x.
The lysine-binding-site-mediated interaction between plasmin and antiplasmin is of great importance for the fast rate of this reaction. It also plays an important part in regulating the fibrinolytic enzyme system. To identify structures important for its noncovalent interaction with plasmin, we constructed seven single-site mutants of antiplasmin by modifying charged amino acids in the C-terminal part of the molecule. All the variants were expressed in the Drosophila S2 cell system, purified, and shown to form stable complexes with plasmin. A kinetic evaluation revealed that two mutants of the C-terminal lysine (K452E or K452T) did not differ significantly from wild-type antiplasmin in their reactions with plasmin, in either the presence or absence of 6-aminohexanoic acid, suggesting that this C-terminal lysine is not important for this reaction. On the other hand, modification of Lys436 to Glu decreased the reaction rate about fivefold compared with wild-type. In addition, in the presence of 6-aminohexanoic acid, only a small decrease in the reaction rate was observed, suggesting that Lys436 is important for the lysine-binding-site-mediated interaction between plasmin and antiplasmin. Results from computerized molecular modelling of the C-terminal 40 amino acids support our experimental data.
纤溶酶与抗纤溶酶之间由赖氨酸结合位点介导的相互作用对于该反应的快速进行极为重要。它在调节纤维蛋白溶解酶系统中也起着重要作用。为了确定其与纤溶酶非共价相互作用的重要结构，我们通过修饰分子C末端部分的带电氨基酸构建了七个抗纤溶酶单点突变体。所有变体均在果蝇S2细胞系统中表达、纯化，并显示能与纤溶酶形成稳定复合物。动力学评估表明，C末端赖氨酸的两个突变体（K452E或K452T）在有或没有6-氨基己酸存在的情况下与纤溶酶的反应与野生型抗纤溶酶没有显著差异，这表明该C末端赖氨酸对该反应并不重要。另一方面，将Lys436突变为Glu后，与野生型相比反应速率降低了约五倍。此外，在6-氨基己酸存在的情况下，仅观察到反应速率有小幅下降，这表明Lys436对于纤溶酶与抗纤溶酶之间由赖氨酸结合位点介导的相互作用很重要。C末端40个氨基酸的计算机分子建模结果支持了我们的实验数据。
