Summary Background Single nucleotide polymorphisms in the vitamin K epoxide reductase (VKOR) gene have been successfully used for warfarin dosage prediction. However, warfarin resistance studies of naturally occurring VKOR mutants do not correlate with their clinical phenotype. This discrepancy presumably arises because the in vitro VKOR activity assay is performed under artificial conditions using the non-physiological reductant dithiothreitol. Objectives The aim of this study is to establish an in vivo VKOR activity assay in mammalian cells(HEK293) where VKOR functions in its native milieu without interference from endogenous enzymes. Methods Endogenous VKOR activity in HEK293 cells was knocked out by transcription activator-like effector nucleases (TALENs)-mediated genome editing. Results and Conclusions Knockout of VKOR in HEK293 cells significantly decreased vitamin K-dependent carboxylation with vitamin K epoxide(KO) as substrate. However, the paralog of VKOR, VKORC1L1, also exhibits substantial ability to convert KO to vitamin K for carboxylation. Using both VKOR and VKORC1L1 knockout cells, we examined the enzymatic activity and warfarin resistance of ten naturally occurring VKOR mutants that were reported previously to have no activity in an in vitro assay. All ten mutants are fully active, five have increased warfarin resistance with the order being W59R>L128R≈W59L>N77S≈S52L. Except for the L128R mutant, this order is consistent with the clinical anticoagulant dosages. The other five VKOR mutants do not change VKOR’s warfarin sensitivity, suggesting that factors other than VKOR play important roles. In addition, we confirmed that the conserved loop cysteines in VKOR are not required for active site regeneration after each cycle of oxidation.

The vitamin K-dependent gamma-glutamyl carboxylase catalyzes the posttranslational modification of select glutamate residues of its vitamin K-dependent substrates to gamma-carboxyglutamate. In this report, we describe a new fluorescence assay that is sensitive and specific for the propeptide binding site of active carboxylase. We employed the assay to make three important observations: (1) A tight binding fluorescein-labeled consensus propeptide can be used to quantify the active fraction of the enzyme. (2) The off-rate for a fluorescein-labeled factor IX propeptide was 3000-fold slower than the rate of carboxylation, a difference that may explain how carboxylase can carry out multiple carboxylations of a substrate during the same binding event. (3) We show evidence that substrate binding to the active site modifies the propeptide binding site of carboxylase. The significant (9-fold) differences in off-rates for the propeptide in the presence and absence of its co-substrates may represent a release mechanism for macromolecular substrates from the enzyme. Additionally, sedimentation velocity and equilibrium experiments indicate a monomeric association of enzyme with propeptide. Furthermore, the carboxylase preparation is monodisperse in the buffer used for our studies.

Patients with mutation L394R in ␥-glutamyl carboxylase have a severe bleeding disorder because of decreased biological activities of all vitamin K-dependent coagulation proteins. Vitamin K administration partially corrects this deficiency. To characterize L394R, we purified recombinant mutant L394R and wild-type carboxylase expressed in baculovirus-infected insect cells. By kinetic studies, we analyzed the catalytic activity of mutant L394R and its binding to factor IX's propeptide and vitamin KH 2 . Mutant L394R differs from its wild-type counterpart as follows: 1) 110-fold higher K i for Boc-mEEV, an active site-specific, competitive inhibitor of FLEEL; 2) 30-fold lower V max /K m toward the substrate FLEEL in the presence of the propeptide; 3) severely reduced activity toward FLEEL carboxylation in the absence of the propeptide; 4) 7-fold decreased affinity for the propeptide; 5) 9-fold higher K m for FIXproGla, a substrate containing the propeptide and the Gla domain of human factor IX; and 6) 5-fold higher K m for vitamin KH 2 . The primary defect in mutant L394R appears to be in its glutamate-binding site. To a lesser degree, the propeptide and KH 2 binding properties are altered in the L394R mutant. Compared with its wildtype counterpart, the L394R mutant shows an augmented activation of FLEEL carboxylation by the propeptide.Vitamin K-dependent carboxylase, also known as ␥-glutamyl carboxylase, an integral membrane protein residing in the rough endoplasmic reticulum, catalyzes the posttranslational modification of specific glutamic acid residues to ␥-carboxyglutamic acid (Gla) 1 in vitamin K-dependent proteins (1). Glacontaining proteins are involved in blood coagulation (2), bone metabolism (3), and regulation of cell proliferation (4). The Gla domains of blood coagulation and anticoagulation proteins mediate calcium-dependent interactions between the protein and phospholipid membranes (5), a process necessary for the biological activity of these proteins. In addition to the glutamate substrate, ␥-glutamyl carboxylation requires carbon dioxide, oxygen, and the essential cofactor vitamin K hydroquinone (KH 2 ), which is the reduced form of vitamin K (6). The formation of Gla from glutamate is coupled with the conversion of vitamin KH 2 to vitamin K 2,3-epoxide. Both of these activities occur in the vitamin K-dependent carboxylase (7, 8). The warfarin-sensitive microsomal enzyme vitamin K epoxide reductase recycles the epoxide back to vitamin KH 2 (9), thus completing the vitamin K cycle.There have been only a few cases of combined deficiencies of vitamin K-dependent coagulation factors reported (10 -18). Patients with this disorder suffer from a bleeding diathesis due to deficiencies of prothrombin and factors VII, IX, and X. In addition, the anticoagulation activities of proteins C and S are decreased. Brenner et al. (16) reported that patients' coagulation activities were severely reduced, while their antigen levels were only moderately decreased. Abnormality of the vitamin K epoxide reductase was rule...

Using reduced vitamin K, oxygen, and carbon dioxide, ␥-glutamyl carboxylase post-translationally modifies certain glutamates by adding carbon dioxide to the ␥ position of those amino acids. In vertebrates, the modification of glutamate residues of target proteins is facilitated by an interaction between a propeptide present on target proteins and the ␥-glutamyl carboxylase. Previously, the gastropod Conus was the only known invertebrate with a demonstrated vitamin K-dependent carboxylase. We report here the discovery of a ␥-glutamyl carboxylase in Drosophila. This Drosophila enzyme is remarkably similar in amino acid sequence to the known mammalian carboxylases; it has 33% sequence identity and 45% sequence similarity to human ␥-glutamyl carboxylase. The Drosophila carboxylase is vitamin K-dependent, and it has a K m toward a model pentapeptide substrate, FLEEL, of about 4 mM. However, unlike the human ␥-glutamyl carboxylase, it is not stimulated by human blood coagulation factor IX propeptides. We found the mRNA for Drosophila ␥-glutamyl carboxylase in virtually every embryonic and adult stage that we investigated, with the highest concentration evident in the adult head.

γ-Glutamyl carboxylase (GC), a polytopic membrane protein found in the endoplasmic reticulum (ER), catalyzes vitamin K–dependent posttranslational modification of glutamate to γ-carboxyl glutamate. In an attempt to delineate the structure of this important enzyme, in vitro translation and in vivo mapping were used to study its membrane topology. Using terminus-tagged full-length carboxylase, expressed in 293 cells, it was demonstrated that the amino-terminus of the GC is on the cytoplasmic side of the ER, while the carboxyl-terminus is on the lumenal side. In addition, a series of fusions were made to encode each predicted transmembrane domain (TMD) followed by a leader peptidase (Lep) reporter tag, as analyzed by the computer algorithm TOPPRED II. Following in vitro translation of each fusion in the presence of canine microsomes, the topological orientation of the Lep tag was determined by proteinase K digestion and endoglycosidase H (Endo H) cleavage. From the topological orientation of the Lep tag in each fusion, the GC spans the ER membrane at least 5 times, with its N-terminus in the cytoplasm and its C-terminus in the lumen.