title: Mutagenesis of vitamin K-dependent carboxylase demonstrates a carboxyl terminus-mediated interaction with vitamin K hydroquinone.
abstract: The gamma-glutamyl carboxylase and vitamin K epoxidase activities of a series of mutants of bovine vitamin K-dependent carboxylase with progressively larger COOH-terminal deletions have been analyzed. The recombinant wild-type (residues 1-758) and mutant protein carboxylases, Cbx 711, Cbx 676, and Cbx 572, representing residues 1-711, 1-676, and 1-572, respectively, were expressed in baculovirus-infected Sf9 cells. Wild-type carboxylase had a Km for the substrate Phe-Leu-Glu-Glu-Leu (FLEEL) of 0.87 mM; the carboxylation of FLEEL was stimulated 2.5-fold by proPT18, the propeptide of prothrombin. Its Km for vitamin K hydroquinone was 23 microM and the specific epoxidase activity of the carboxylase was 938 pmol vitamin KO/30 min/pmol of carboxylase. Cbx 711, which was also stimulated by proPT18, had a Km for FLEEL, a Km for vitamin K hydroquinone, and a specific epoxidase activity that was comparable to the wild-type carboxylase. In contrast Cbx 572 lacked both carboxylase and epoxidase activities. Although Cbx 676 had a normal carboxylase active site in terms of the Km for FLEEL and its stimulation by proPT18, the Km for vitamin K hydroquinone was 540 microM, and the specific epoxidase activity was 97 pmol KO/30 min/pmol of Cbx 676. The catalytic efficiencies of Cbx 676 for glutamate carboxylation and vitamin K epoxidation were decreased 15- and 400-fold, respectively, from wild-type enzyme reflecting the requirement for formation of an activated vitamin K species for carboxylation to occur. These data indicate that the truncation of COOH-terminal segments of the carboxylase had no effect on FLEEL or propeptide recognition, but in the case of Cbx 676, selectively affected the interaction with vitamin K hydroquinone and the generation of epoxidase activity. These data suggest that a vitamin K epoxidase activity domain may reside near the COOH terminus while the carboxylase active site domain resides toward the NH2 terminus.
authors.affiliation: Center for Hemostasis and Thrombosis Research, New England Medical Center, Boston, Massachusetts.
authors.forename: D A
authors.initials: DA
authors.lastname: Roth
authors.forename: M L
authors.initials: ML
authors.lastname: Whirl
authors.forename: L J
authors.initials: LJ
authors.lastname: Velazquez-Estades
authors.forename: C T
authors.initials: CT
authors.lastname: Walsh
authors.forename: B
authors.initials: B
authors.lastname: Furie
authors.forename: B C
authors.initials: BC
authors.lastname: Furie
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D019735:Carbon-Carbon Ligases; D002417:Cattle; D002460:Cell Line; D003062:Codon; D007700:Kinetics; D008025:Ligases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D018411:Spodoptera; D013379:Substrate Specificity; D014162:Transfection; D010837:Vitamin K 1 / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D003062:Codon; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C028843:vitamin K1 hydroquinone; D010837:Vitamin K 1; D008025:Ligases; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase