tailieunhanh - Báo cáo khoa học: Identification of amino acids in antiplasmin involved in its noncovalent ‘lysine-binding-site’-dependent interaction with plasmin

The lysine-binding-site-mediated interaction between plas-min and antiplasmin is of great importance for the fast rate of this reaction. It also plays an important part in regulat-ing 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 theC-terminal part of the molecule. | Eur. J. Biochem. 270 2023-2029 2003 FEBS 2003 oi 5 2-l 2 iOiO 0 5 Identification of amino acids in antiplasmin involved in its noncovalent lysine-binding-site -dependent interaction with plasmin Haiyao Wang Anna Yu Bjorn Wiman and Sarolta Pap Department of Clinical Chemistry and Blood Coagulation Karolinska Hospital Karolinska Institute Stockholm Sweden 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. Keywords antiplasmin fibrinolysis lysine-binding site mutagenesis plasmin. The reaction between plasmin and its natural inhibitor in blood plasma antiplasmin normally occurs in several sequential steps --4 . The first step which is rate limiting takes place between one of the so called lysine-binding sites in the plasmin .