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.

Profactor IX Propeptide and Glutamate Substrate Binding Sites on the Vitamin K-dependent Carboxylase Identified by Site-directed Mutagenesis

“…Vitamin K epoxide formation was deter-mined as described previously (21). Briefly, upon completion of the 30-min incubation at 25°C in sealed tubes, the reaction mixture was extracted with 250 l of ethanol and then 750 l of hexane.…”

“…We have recently localized the binding site of the carboxylase for the factor IX propeptide (19) and a small carboxylatable peptide substrate (20) to the hydrophobic region of the enzyme using affinity-labeling reagents. We have also shown that epoxidase activity is affected by truncation of the C terminus of the enzyme, a modification that does not alter binding of propeptide or a carboxylatable peptide substrate (21).…”

Propeptide and glutamate-containing substrates bound to the vitamin K-dependent carboxylase convert its vitamin K epoxidase function from an inactive to an active state

“…Epoxidase assays were performed in a 125-l mixture as described above except that NaH 14 CO 3 was replaced with the same concentration of NaHCO 3 . Vitamin K epoxide formation was determined as described (24). Briefly, upon completion of the 30-min incubation at 25°C in sealed tubes, the reaction mixture was extracted with 250 l of ethanol and then with 750 l of hexane.…”

The Vitamin K-dependent Carboxylase Has Been Acquired by Leptospira Pathogens and Shows Altered Activity That Suggests a Role Other than Protein Carboxylation

“…all of the residues downstream of Pro-756 in Fig. 2) resulted in a decrease in both catalytic efficiency toward vitamin K hydroquinone and epoxidase activity, which led to the proposal that the vitamin K epoxidase domain may reside near the C terminus (30). However, this region is not present in the Leptospira enzyme (Fig.…”