Trimeresurus stejnegeri snake venom plasminogen activator. Site-directed mutagenesis and molecular modeling.
The specific plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) is a serine proteinase presenting 23% sequence identity with the proteinase domain of tissue type plasminogen activator, and 63% with batroxobin, a fibrinogen clotting enzyme from Bothrops atrox venom that does not activate plasminogen. TSV-PA contains six disulfide bonds and has been successfully overexpressed in Escherichia coli (Zhang, Y., Wisner, A., Xiong, Y. L., and Bon, C. (1995) J. Biol. Chem. 270, 10246-10255). To identify the functional domains of TSV-PA, we focused on three short peptide fragments of TSV-PA showing important sequence differences with batroxobin and other venom serine proteinases. Molecular modeling shows that these sequences are located in surface loop regions, one of which is next to the catalytic site. When these sequences were replaced in TSV-PA by the equivalent batroxobin residues none generated either fibrinogen-clotting or direct fibrinogenolytic activity. Two of the replacements had little effect in general and are not critical to the specificity of TSV-PA for plasminogen. Nevertheless, the third replacement, produced by the conversion of the sequence DDE 96a-98 to NVI, significantly increased the Km for some tripeptide chromogenic substrates and resulted in undetectable plasminogen activation, indicating the key role that the sequence plays in substrate recognition by the enzyme.
Zhang
Y
Y
Unité des Venins, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France.
Wisner
A
A
Maroun
R C
RC
Choumet
V
V
Xiong
Y
Y
Bon
C
C
D000595:Amino Acid Sequence; D003435:Crotalid Venoms / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010960:Plasminogen Activators / Q000096:biosynthesis / Q000737:chemistry*; D011994:Recombinant Proteins / Q000096:biosynthesis
D003435:Crotalid Venoms; D011994:Recombinant Proteins; D010960:Plasminogen Activators