MUTAGENESIS OF VITAMIN-K-DEPENDENT CARBOXYLASE DEMONSTRATES A CARBOXYL TERMINUS-MEDIATED INTERACTION WITH VITAMIN-K HYDROQUINONE

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 K-m 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 K-m for vitamin K hydroquinone was 23 mu M 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 K-m for FLEEL, a K-m 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 K-m for FLEEL and its stimulation by proPT18, the K-m for vitamin K hydroquinone was 540 mu M, 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.