Construction and Characterization of a Mutant of Single-chain Urokinase-type Plasminogen Activator Ser(175)-His(187)-mscu-PA
Single-chain urokinase-type plasminogen activator scu-PA is the precursor of double-chain urokinase tcu-PA , which has a much higher intrinsic catalytic activity than other zymogens of the serine protease family. To restore the "zymogen triad" of Asp-His-Ser in the serine protease family, the mutant gene of scu-PA mscu-PA, Ala(175)right curved arrow Ser(175), Tyr(187)right curved arrow His(187) was constructed by the method of oligonucleotide-directed, site-specific mutagenesis in order to reduce its intrinsic catalytic activity. mscu-PA was expressed in E. coli BL21. After denaturation and renaturation in vitro, the mscu-PA was purified to homogeneity by SP-Sepharose ion-exchange chromatography, Sephacryl S-200 chromatography and Benzamidine-Sepharose affinity adsorption. mscu-PA had the same activity to plasmin as scu-PA. The catalytic efficiency measured by k(cat)/K(m) to synthetic substrate S(2444) was 2.5-fold lower than that of scu-PA, and the activity against Glu-plasminogen was also reduced. After activation by plasmin, mtcu-PA and tcu-PA had similar catalytic efficiency against S(2444) and Glu-plasminogen. The results suggest that the intrinsic catalytic activity of mscu-PA be really reduced after restoring the "zymogen triad".
Department of Biochemistry, National Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, China. zma@netra.nju.edu.cn
Yu-Ming
YM
Xue
Hui
H
Zhu
Wei
W
Shi
Wei
W
Liu
Jian-Ning
JN
Liu
Zhong
Z
Ma