Mutagenesis at Pro309 of single-chain urokinase-type plasminogen activator alters its catalytic properties.
The charge of Lys300(c143) located within a flexible loop(297-313) of sc-uPA has been identified as an important determinant for its high intrinsic activity. Mutations affecting the flexibility of the loop also modulate the intrinsic activity. Glu-plasminogen activation by sc-uPA is strongly promoted by fibrin fragment E but not fibrin fragment D-dimer, whereas plasminogen activation by t-PA is strongly promoted by fragment D-dimer but not fragment E. To further investigate the effect of conformation changes in the flexible loop on catalytic properties of sc-uPA, cassette mutations at Pro309(c152) were made and characterized. It was found that the activation of Pro309(c152) mutants by Lys-plasmin was only moderately affected. In contrast, the intrinsic and two-chain activities of Pro309(c152) mutants against S2444 were both significantly decreased. The two-chain activities of these mutants against Glu-plasminogen were also reduced in a range of 1.1- to 127-fold. The mutations of Pro309(c152) to Trp/Phe and Arg/Asp more significantly affected both intrinsic and two-chain activities, while only a moderate decrease in activity was found with mutations to Ala/Ser/Thr. In contrast to wild-type sc-uPA, plasminogen activation by Pro309(c152) mutants was found to be promoted by both fibrin fragment E and D-dimer. In the presence of 2.0 microM D-dimer, plasminogen activation by mutant Pro309(c152) --> His was promoted by 22-fold, while only 2.0-fold promotion was found with mutant Pro309(c152) --> Gly. In conclusion, these findings demonstrated that conformation changes in the flexible loop of sc-uPA not only affect its intrinsic and two-chain activity, but also extend its promotion of plasminogen activation by fragment E to D-dimer.
Sun
Ziyong
Z
Institute of Molecular Medicine, State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, China.
Liu
Jian-Ning
JN
D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010958:Plasminogen / Q000737:chemistry / Q000378:metabolism*; D011392:Proline; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D018076:DNA, Complementary; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010958:Plasminogen; D011392:Proline; D014568:Urokinase-Type Plasminogen Activator