tailieunhanh - Báo cáo khoa học: Limited mutagenesis increases the stability of human carboxypeptidase U (TAFIa) and demonstrates the importance of CPU stability over proCPU concentration in down-regulating fibrinolysis

Procarboxypeptidase U [proCPU, thrombin-activatable fibrinolysis inhib-itor (TAFI), EC ] 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. | iFEBS Journal Limited mutagenesis increases the stability of human carboxypeptidase U TAFIa and demonstrates the importance of CPU stability over proCPU concentration in down-regulating fibrinolysis Wolfgang Knecht1 Johan Willemse3 Hanna Stenhamre1 Mats Andersson2 Pia Berntsson1 Christina Furebring2 Anna Harrysson1 Ann-Christin Malmborg Hager2 Britt-Marie Wissing1 Dirk Hendriks3 and Philippe Cronet1 1 AstraZeneca R D Molndal Molndal Sweden 2 Alligator Bioscience AB Lund Sweden 3 Laboratory of MedicalBiochemistry University of Antwerp Wilrijk Belgium Keywords carboxypeptidase coagulation directed evolution fibrinolysis protease Correspondence W. Knecht Molecular Pharmacology -Target Production AstraZeneca R D Molndal 431 83 Molndal Sweden Fax 46 317763753 Tel 46 317065341 E-mail Received 5 November 2005 accepted 19 December 2005 doi Procarboxypeptidase U proCPU thrombin-activatable fibrinolysis inhibitor TAFI EC 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 h of wild-type CPU to h for the mutants. We provide evidence that the gain in stable activity is accompanied by a gain .