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Limited mutagenesis increases the stability of human carboxypeptidase U (TAFIa) and demonstrates the importance of CPU stability over proCPU concentration in down‐regulating fibrinolysis
Author(s) -
Knecht Wolfgang,
Willemse Johan,
Stenhamre Hanna,
Andersson Mats,
Berntsson Pia,
Furebring Christina,
Harrysson Anna,
Hager AnnChristin Malmborg,
Wissing BrittMarie,
Hendriks Dirk,
Cronet Philippe
Publication year - 2006
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2006.05110.x
Subject(s) - carboxypeptidase , fibrinolysis , chemistry , mutagenesis , zymogen , central processing unit , mutant , enzyme , biochemistry , computer science , medicine , gene , operating system
Procarboxypeptidase U [proCPU, thrombin‐activatable fibrinolysis inhibitor (TAFI), EC 3.4.17.20] belongs to the metallocarboxypeptidase family and is a zymogen found in human plasma. ProCPU has been proposed to be a molecular link between coagulation and fibrinolysis. Upon activation of proCPU, the active enzyme (CPU) rapidly becomes inactive due to its intrinsic instability. The inherent instability of CPU is likely to be of major importance for the in vivo down‐regulation of its activity, but the underlying structural mechanisms of this fast and spontaneous loss of activity of CPU have not yet been explained, and they severely inhibit the structural characterization of CPU. In this study, we screened for more thermostable versions of CPU to increase our understanding of the mechanism underlying the instability of CPU's activity. We have shown that single as well as a few 2–4 mutations in human CPU can prolong the half‐life of CPU's activity at 37 °C from 0.2 h of wild‐type CPU to 0.5–5.5 h for the mutants. We provide evidence that the gain in stable activity is accompanied by a gain in thermostability of the enzyme and increased resistance to proteolytic digest by trypsin. Using one of the stable mutants, we demonstrate the importance of CPU stability over proCPU concentration in down‐regulating fibrinolysis.