Identification of amino acids in antiplasmin involved in its noncovalent 'lysine-binding-site'-dependent interaction with plasmin.
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.
Wang
Haiyao
H
Department of Clinical Chemistry and Blood Coagulation, Karolinska Hospital, Karolinska Institute, Stockholm, Sweden.
Yu
Anna
A
Wiman
Björn
B
Pap
Sarolta
S
D000595:Amino Acid Sequence; D000818:Animals; D000933:Antifibrinolytic Agents / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D003198:Computer Simulation; D004331:Drosophila melanogaster / Q000502:physiology; D005341:Fibrinolysin / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000979:alpha-2-Antiplasmin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000933:Antifibrinolytic Agents; D020033:Protein Isoforms; D015842:Serine Proteinase Inhibitors; D000979:alpha-2-Antiplasmin; D005341:Fibrinolysin; D008239:Lysine