Implication of cysteine residues in the activity of single-chain urokinase-plasminogen activator.
Single-chain urokinase-plasminogen activator contains 24 cysteine residues involved in 12 disulfide bonds and distributed all along the three domains of the protein. In order to investigate the role of these disulfide bridges in the catalytic activities of scu-PA, we used site-specific mutagenesis to construct 10 mutants in which some cysteine residues were changed to serine residues. Each mutated DNA fragment was cloned into a procaryotic expression vector and the protein expressed in E. coli. Mutant proteins of the expected size were produced and analyzed for amidolytic and fibrinolytic activities. From this, it is shown that: i) the disulfide bonds in the epidermal growth factor (EGF)-like and in the kringle domains are not necessary. Moreover, disulfide bond deletion in the kringle domain improves those catalytic activities; ii) on the contrary, the disulfide bridges in the catalytic domain are essential for maintaining both activities.
Hamelin
J
J
Centre Hayem, Hôpital Saint-Louis, Paris, France.
Sarmientos
P
P
Orsini
G
G
Galibert
F
F
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003545:Cysteine; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005337:Fibrin / Q000378:metabolism; D005342:Fibrinolysis; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D004220:Disulfides; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D005337:Fibrin; D014568:Urokinase-Type Plasminogen Activator; D003545:Cysteine