Construction and
Characterization of a Mutant of Single-chain Urokinase-type Plasminogen
Activator （Ser175-His187-mscu-PA）
XUE Yu-Ming, ZHU Hui, SHI Wei, LIU Wei, LIU Jian-Ning, MA
Zhong*
(Department of Biochemistry, National Key Laboratory of Pharmaceutical
Biotechnology,
Nanjing University, Nanjing 210093, China)
Abstract 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, Ala175→Ser175, Tyr187→His187） 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 kcat/Km） to synthetic substrate
S2444 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 S2444 and Glu-plasminogen. The
results suggest that the intrinsic catalytic activity of mscu-PA be really
reduced after restoring the “zymogen triad”.
Key words single chain urokinase-type
plasminogen activator；
site-specific mutagenesis； intrinsic catalytic
activity；
zymogen triad
Received: May 31,
1999 Accepted: August 19, 1999
* Corresponding author: Tel, 86-25-3592331; Fax, 86-25-3593796; e-mail, [email protected]