Vitamin K epoxide reductase (VKOR) is the target of warfarin, the most widely prescribed anticoagulant for thromboembolic disorders. Although estimated to prevent twenty strokes per induced bleeding episode, warfarin is under-used because of the difficulty of controlling dosage and the fear of inducing bleeding. Although identified in 1974 (ref. 2), the enzyme has yet to be purified or its gene identified. A positional cloning approach has become possible after the mapping of warfarin resistance to rat chromosome 1 (ref. 3) and of vitamin K-dependent protein deficiencies to the syntenic region of human chromosome 16 (ref. 4). Localization of VKOR to 190 genes within human chromosome 16p12-q21 narrowed the search to 13 genes encoding candidate transmembrane proteins, and we used short interfering RNA (siRNA) pools against individual genes to test their ability to inhibit VKOR activity in human cells. Here, we report the identification of the gene for VKOR based on specific inhibition of VKOR activity by a single siRNA pool. We confirmed that MGC11276 messenger RNA encodes VKOR through its expression in insect cells and sensitivity to warfarin. The expressed enzyme is 163 amino acids long, with at least one transmembrane domain. Identification of the VKOR gene extends our understanding of blood clotting, and should facilitate development of new anticoagulant drugs.

The vitamin K-dependent ␥-glutamyl carboxylase catalyzes the modification of specific glutamates in a number of proteins required for blood coagulation and associated with bone and calcium homeostasis. All known vitamin K-dependent proteins possess a conserved eighteen-amino acid propeptide sequence that is the primary binding site for the carboxylase. We compared the relative affinities of synthetic propeptides of nine human vitamin K-dependent proteins by determining the inhibition constants (K i ) toward a factor IX propeptide/ ␥-carboxyglutamic acid domain substrate. The K i values for six of the propeptides (factor X, matrix Gla protein, factor VII, factor IX, PRGP1, and protein S) were between 2-35 nM, with the factor X propeptide having the tightest affinity. In contrast, the inhibition constants for the propeptides of prothrombin and protein C are ϳ100-fold weaker than the factor X propeptide. The propeptide of bone Gla protein demonstrates severely impaired carboxylase binding with an inhibition constant of at least 200,000-fold weaker than the factor X propeptide. This study demonstrates that the affinities of the propeptides of the vitamin K-dependent proteins vary over a considerable range; this may have important physiological consequences in the levels of vitamin Kdependent proteins and the biochemical mechanism by which these substrates are modified by the carboxylase.The vitamin K-dependent carboxylase catalyzes the posttranslational modification of specific glutamates to ␥-carboxyglutamate (Gla) 1 in a number of proteins. Most vitamin K-dependent proteins are involved in the hemostatic process (prothrombin, factors VII, IX, and X, and proteins C, S, and Z), whereas two others (bone Gla protein and matrix Gla protein) are associated with bone (1-4). Two new putative vitamin K-dependent proteins of unassigned function, proline-rich Gla proteins (PRGP1 and PRGP2), were identified by sequence homology searches and are believed to be membrane proteins (5).A conserved eighteen-amino acid sequence essential for substrate recognition is found in all vitamin K-dependent proteins and was first identified by Pan and Price (6) based on sequence comparisons of the blood and bone vitamin K-dependent proteins. The conserved region is present as a propeptide sequence amino-terminal to the highly conserved Gla domains of the vitamin K-dependent blood proteins and is proteolytically removed to form the mature protein. With bone Gla protein, this sequence is also present as a propeptide amino-terminal to the mature form of the protein, whereas with matrix Gla protein the vitamin K-dependent propeptide-like sequence is part of the mature form of the protein (7). Confirmation of the importance of the propeptide sequence in carboxylation is demonstrated by experiments where deletion of the propeptide abrogates carboxylation of factor IX or protein C expressed in cell culture (8, 9). In addition, mutagenesis studies have identified a number of highly conserved amino acids (e.g. Phe Ϫ16, Ala Ϫ10, Leu Ϫ6) as well as les...

Interaction between L17 in the ribosome tunnel and folded nascent chain transmembrane segments during multi-spanning membrane protein synthesis triggers structural rearrangements in the ribosome that cause switching between cytosolic and ER lumenal targeting of the growing polypeptide.

The vitamin K-dependent ␥-glutamyl carboxylase binds an 18-amino acid sequence usually attached as a propeptide to its substrates. Price and Williamson (Protein Sci. (1993) 2, 1997-1998) noticed that residues 495-513 of the carboxylase shares similarity with the propeptide. They suggested that this internal propeptide could bind intramolecularly to the propeptide binding site of carboxylase, thereby preventing carboxylation of substrates lacking a propeptide recognition sequence. To test Price's hypothesis, we created nine mutant enzyme species that have single or double mutations within this putative internal propeptide. The apparent K d values of these mutant enzymes for human factor IX propeptide varied from 0.5-to 287-fold when compared with that of wild type enzyme. These results are consistent with the internal propeptide hypothesis but could also be explained by these residues participating in propeptide binding site per se. To distinguish between the two alternative hypotheses, we measured the dissociation rates of propeptides from each of the mutant enzymes. Changes in an internal propeptide should not affect the dissociation rates, but changes to a propeptide binding site may affect the dissociation rate. We found that dissociation rates varied in a manner consistent with the apparent K d values measured above. Furthermore, kinetic studies using propeptide-containing substrates demonstrated a correlation between the affinity for propeptide and V max . Taken together, our results indicated that these mutations affected the propeptide binding site rather than a competitive inhibitory internal propeptide sequence. These results agree with our previous observations, indicating that residues in this region are involved in propeptide binding.The vitamin K-dependent ␥-glutamyl carboxylase is a polytopic integral membrane protein that resides in the endoplasmic reticulum (1). It catalyzes the post-translational modification of a number of vitamin K-dependent proteins (e.g. the coagulation proteins prothrombin, factor VII, factor IX, factor X, protein S, protein C, and protein Z) (2, 3). Other known vitamin K-dependent proteins are the bone-related proteins osteocalcin and matrix Gla protein, the growth arrest protein Gas 6, and four proteins of unknown function: proline-rich Gla proteins I and II and TMG proteins 3 and 4 (4 -9). Vitamin K-dependent carboxylase utilizes the substrates: reduced vitamin K, carbon dioxide, oxygen, and a propeptide-containing substrate. Multiple glutamic acid residues of the polypeptide substrate, within about 40 residues of the propeptide, are usually modified to ␥-carboxyglutamate during a single binding event (10).The primary interaction between the vitamin K-dependent carboxylase and its substrates is mediated by the 18-amino acid propeptide sequence (11, 12), which in all known vitamin K-dependent proteins, except for matrix Gla protein, is removed prior to secretion. The role of the substrate's propeptide is to anchor it to the carboxylase for a time sufficient for multiple c...

The ␥-glutamyl carboxylase utilizes four substrates to catalyze carboxylation of certain glutamic acid residues in vitamin K-dependent proteins. How the enzyme brings the substrates together to promote catalysis is an important question in understanding the structure and function of this enzyme. The propeptide is the primary binding site of the vitamin K-dependent proteins to carboxylase. It is also an effector of carboxylase activity. We tested the hypothesis that binding of substrates causes changes to the carboxylase and in turn to the substrate-enzyme interactions. In addition we investigated how the sequences of the propeptides affected the substrate-enzyme interaction. To study these questions we employed fluorescently labeled propeptides to measure affinity for the carboxylase. We also measured the ability of several propeptides to increase carboxylase catalytic activity. Finally we determined the effect of substrates: vitamin K hydroquinone, the pentapeptide FLEEL, and NaHCO 3 , on the stability of the propeptide-carboxylase complexes. We found a wide variation in the propeptide affinities for carboxylase. In contrast, the propeptides tested had similar effects on carboxylase catalytic activity. FLEEL and vitamin K hydroquinone both stabilized the propeptide-carboxylase complex. The two together had a greater effect than either alone. We conclude that the effect of propeptide and substrates on carboxylase controls the order of substrate binding in such a way as to ensure efficient, specific carboxylation.The vitamin K-dependent ␥-glutamyl carboxylase is an endoplasmic reticulum integral membrane enzyme. It post-translationally modifies certain proteins important in several physiologically important areas, including blood coagulation. The modification involves the addition of a carboxyl group to the ␥-carbon of glutamic acid residues in the amino portion of the vitamin K-dependent substrate.In addition to the glutamic acid and CO 2 , which provides the added carboxyl group, other substrates required for the reaction are vitamin K hydroquinone (KH 2 ) 4 and oxygen. During carboxylation KH 2 is converted to the epoxide; thus carboxylase is also an epoxidase.The primary interaction between carboxylase and most of its protein substrates is through the propeptides of the substrates. Binding is of high affinity, which allows the substrate to bind long enough for multiple carboxylations to occur.In addition to being the primary binding site for carboxylase, the propeptide also exerts an effect on carboxylase catalysis. Factors IX and X and prothrombin propeptides, when bound to carboxylase, increase the activity of the enzyme toward small glutamate-containing substrates (1-5).For the most part these results suggest that the propeptides induce a conformational change or stabilize a more active conformation in the carboxylase active site. The efficiency of the change appears to be similar for the propeptides of prothrombin and factors IX and X. The fact that these propeptides have different sequences implies that ...

Summary. A mutation (W501S) in the vitamin K-dependent c-glutamyl carboxylase (VKC) that leads to a congenital bleeding disorder was recently discovered in two patients. To characterize the enzyme defect, recombinant VKC-W501S was expressed in and purified from insect cells. The major effect of the mutation appears to be to decrease the affinity of the carboxylase for the propeptide of its substrates. This observation agrees with recent data that place part of the propeptide binding site within residues 495-513 of VKC. Additionally, we demonstrate that the affinity between descarboxy osteocalcin (d-OC) and VKC remains unaffected by the W501S mutation. This confirms earlier data that the high-affinity site for d-OC is not located on the propeptide binding domain of VKC. Two properties of the enzyme suggest an explanation for the observation that vitamin K supplementation ameliorates the effects of the mutation: (i) since full carboxylation requires the propeptide to remain bound to the enzyme sufficiently long for full carboxylation, a reduced affinity can cause its premature release before carboxylation is complete; (ii) propeptide binding results in a decrease of the K M for vitamin K hydroquinone in wild-type, but not in mutant carboxylase, resulting in increased vitamin K requirement of affected subjects.

Propeptides of the vitamin K-dependent proteins bind to an exosite on ␥-glutamyl carboxylase; while they are bound, multiple glutamic acids in the ␥-carboxyglutamic acid (Gla) domain are carboxylated. The role of the propeptides has been studied extensively; however, the role of the Gla domain in substrate binding is less well understood. We used kinetic and fluorescence techniques to investigate the interactions of the carboxylase with a substrate containing the propeptide and Gla domain of factor IX (FIXproGla41 2) The Gla domain plays an allosteric role in substrate-enzyme interactions. 3) Carboxylation reduces the allosteric effect. 4) The similarity between the steady state carboxylation rate constant and product dissociation rate constant suggests that product release is rate-limiting. 5) The increased dissociation rate after carboxylation contributes to the release of product.The vitamin K-dependent ␥-glutamyl carboxylase is an integral membrane protein located in the endoplasmic reticulum. It catalyzes the post-translational modification of specific glutamic acid residues to ␥-carboxylglutamic acid in a number of vitamin K-dependent proteins. In these vitamin K-dependent proteins, multiple glutamic acid residues in the amino-terminal Gla 1 domain are modified (1-3). The existence of these multiple ␥-carboxylglutamic acid residues allows the Gla domain to form the calcium-dependent conformation required for the activity of these vitamin K-dependent proteins (4, 5). Under-carboxylated vitamin K-dependent proteins cannot form this calcium-dependent structure and, as a result, possess poor affinities for phospholipid surfaces, endothelial cells, or activated platelets (6 -8).Previous studies indicate that all or nearly all of the Glu residues to be carboxylated are modified during a single substrate binding event (9, 10). The loss of as few as three carboxylations can markedly decrease the activities of vitamin K-dependent proteins (8). Therefore, because the most frequently used anticoagulant, warfarin, causes under-carboxylation by reducing vitamin K concentration, understanding the mechanism by which processivity yields functional enzymes is important.A tethered model has been proposed to account for how carboxylase accomplishes full carboxylation of pro-factor IX (9, 11). In this hypothesis, the primary interaction between carboxylase and its substrates is mediated by a propeptide sequence of 18 amino acids. Presumably, the propeptide anchors the Gla domain of the substrate near the carboxylase active site for a time sufficient for modification of all or most of the Glu residues of the Gla domain.Several lines of evidence are consistent with the idea that full carboxylation is the result of a stochastic, processive mechanism, i.e. the degree of carboxylation is controlled by the balance between the carboxylation rate of the substrate and the dissociation rate of the product. First, bone Gla protein, whose propeptide has an extremely low affinity (K i Ͼ 500 M) for the carboxylase and therefore a rapid dis...

Antibodies to Epstein-Barr virus capsid antigen (anti-VCA) and early antigen (anti-EA) were measured in 263 patients with nasopharyngeal carcinoma (NPC), 624 age- and sex-matched neighborhood controls, 570 family members of NPC patients and 830 family members of neighborhood controls in Taiwan. The distribution of antibody titers was significantly different between NPC patients and the other three groups. More than 55% and 45% of NPC patients had titers of greater than or equal to 1:640 and greater than or equal to 1:80 for anti-VCA and anti-EA, respectively, while less than 6.7% and 2.5% of the other three groups had such high titers. The geometric means of anti-VCA and anti-EA titers were 1:352 and 1:45, respectively, in NPC patients compared to less than 1:77 and 1:12, respectively, in the comparison groups. Anti-VCA and anti-EA titers were significantly correlated. The association of EBV with NPC is discussed.