title: Mutations in the substrate binding site of thrombin-activatable fibrinolysis inhibitor (TAFI) alter its substrate specificity.
abstract: Thrombin-activable fibrinolysis inhibitor (TAFI) is a zymogen that inhibits the amplification of plasmin production when converted to its active form (TAFIa). TAFI is structurally very similar to pancreatic procarboxypeptidase B. TAFI also shares high homology in zinc binding and catalytic sites with the second basic carboxypeptidase present in plasma, carboxypeptidase N. We investigated the effects of altering residues involved in substrate specificity to understand how they contribute to the enzymatic differences between TAFI and carboxypeptidase N. We expressed wild type TAFI and binding site mutants in 293 cells. Recombinant proteins were purified and characterized for their activation and enzymatic activity as well as functional activity. Although the thrombin/thrombomodulin complex activated all the mutants, carboxypeptidase B activity of the activated mutants against hippuryl-arginine was reduced. Potato carboxypeptidase inhibitor inhibited the residual activity of the mutants. The functional activity of the mutants in a plasma clot lysis assay correlated with their chromogenic activity. The effect of the mutations on other substrates depended on the particular mutation, with some of the mutants possessing more activity against hippuryl-His-leucine than wild type TAFIa. Thus mutations in residues around the substrate binding site of TAFI resulted in altered C-terminal substrate specificity.
authors.affiliation: Department of Cardiovascular Research, Berlex Biosciences, Richmond, California 94806,USA. lei_zhao@berlex.com
authors.forename: Lei
authors.initials: L
authors.lastname: Zhao
authors.forename: Brad
authors.initials: B
authors.lastname: Buckman
authors.forename: Marian
authors.initials: M
authors.lastname: Seto
authors.forename: John
authors.initials: J
authors.lastname: Morser
authors.forename: Mariko
authors.initials: M
authors.lastname: Nagashima
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D025901:Carboxypeptidase B2 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002268:Carboxypeptidases / Q000378:metabolism; D017931:DNA Primers; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D002268:Carboxypeptidases; D025901:Carboxypeptidase B2