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.
Highlights
° activity is under intense study, A recently proposed basicity enhancement model (Ham and Dowd, 1990) supported by 18 2_ labeling experiments (Dowd et al, 1992; Kuliopulos et al, 1992b) proposes that CO2 fixation is a late-stage reactionterminating event which follows 02-dependent enzyme catalyzed formation of vitamin K 2,3-epoxide from vitamin K hydroquinone
The recent affinity purification of bovine liver carboxylase (Wu et al, 1991b; Kuliopulos et al, 1992a) has lead to cloning the full-length human liver cDNA (Wu et al, 1991a) and the full-length bovine liver cDNA (Rehemtulla et al, 1993). While expression of these cDNAs in heterologous mammalian cells results in an increase in carboxylase activity above endogenous background levels (Wu et al, 1991a; Rehemtulla et al, 1993), the expression of the bovine liver cDNA in baculovirus-infected insect cells has been useful due to the lack of endogenous vitamin K-dependent carboxylase activity in these cells (Roth et al, 1993)
The plasmid pBLIIlbCbx was digested with NheI and HindUI, and the fragment containing the carboxylase coding sequence was directionally subcloned into the isolated mutagenesis phagemid vector pALTER-1 which had been linearized with XbaI and HindU!
Summary
Vol 270, No 10, Issue of March 10, pp. 5305-5311, 1995 Printed in U.S.A. Mutagenesis of Vitamin K-dependent Carboxylase Demonstrates a Carboxyl Terminus-mediated Interaction with Vitamin K Hydroquinone*. 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. Site-directed mutagenesis was pursued to create a series of mutant enzymes with progressively larger deletions of the carboxyl terminus Characterization of these mutant proteins, expressed in baculovirus-infected insect cells, provides the first functional evidence to suggest a role for the carboxyl terminus in mediating an interaction with vitamin K hydroquinone
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