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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.
Limited mutagenesis increases the stability of human carboxypeptidase U (TAFIa) and demonstrates the importance of CPU stability over proCPU concentration in down-regulating fibrinolysis.
- Source :
-
The FEBS journal [FEBS J] 2006 Feb; Vol. 273 (4), pp. 778-92. - Publication Year :
- 2006
-
Abstract
- 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 degrees 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.
- Subjects :
- Amino Acid Sequence
Animals
Blood Coagulation
Carboxypeptidase B2 chemistry
Carboxypeptidase B2 genetics
Cell Line
Down-Regulation
Enzyme Activation
Enzyme Stability
Fibrin genetics
Fibrin metabolism
Hot Temperature
Humans
Lysine metabolism
Molecular Sequence Data
Point Mutation
Protein Denaturation
Protein Precursors chemistry
Protein Precursors genetics
Sequence Alignment
Carboxypeptidase B2 metabolism
Fibrinolysis
Mutagenesis
Protein Precursors metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1742-464X
- Volume :
- 273
- Issue :
- 4
- Database :
- MEDLINE
- Journal :
- The FEBS journal
- Publication Type :
- Academic Journal
- Accession number :
- 16441664
- Full Text :
- https://doi.org/10.1111/j.1742-4658.2006.05110.x