The Vitamin K Oxidoreductase Is a Multimer That Efficiently Reduces Vitamin K Epoxide to Hydroquinone to Allow Vitamin K-dependent Protein Carboxylation

The vitamin K oxidoreductase (VKORC1) recycles vitamin K to support the activation of vitamin K-dependent (VKD) proteins, which have diverse functions that include hemostasis and calcification. VKD proteins are activated by Glu carboxylation, which depends upon the oxygenation of vitamin K hydroquinone (KH₂). The vitamin K epoxide (KO) product is recycled by two reactions, i.e. KO reduction to vitamin K quinone (K) and then to KH₂, and recent studies have called into question whether VKORC1 reduces K to KH₂. Analysis in insect cells lacking endogenous carboxylation components showed that r-VKORC1 reduces KO to efficiently drive carboxylation, indicating KH₂ production. Direct detection of the vitamin K reaction products is confounded by KH₂ oxidation, and we therefore developed a new assay that stabilized KH₂ and allowed quantitation. Purified VKORC1 analyzed in this assay showed efficient KO to KH₂ reduction. Studies in 293 cells expressing tagged r-VKORC1 revealed that VKORC1 is a multimer, most likely a dimer. A monomer can only perform one reaction, and a dimer is therefore interesting in explaining how VKORC1 accomplishes both reactions. An inactive mutant (VKORC1(C132A/C135A)) was dominant negative in heterodimers with wild type VKORC1, resulting in decreased KO reduction in cells and carboxylation in vitro. The results are significant regarding human VKORC1 mutations, as warfarin-resistant patients have mutant and wild type VKORC1 alleles. A VKORC1 dimer indicates a mixed population of homodimers and heterodimers that may have different functional properties, and VKORC1 reduction may therefore be more complex in these patients than appreciated previously.     

Synthesis of menaquinone-2 derivatives as substrates for the liver microsomal vitamin K-dependent carboxylase

The rat liver microsomal vitamin K-dependent carboxylase catalyzes the carboxylation of glutamyl to gamma-carboxyglutamyl residues in the presence of reduced vitamin K, O2 and CO2. The specificity of the enzyme for the vitamin substrate has been probed by the synthesis of a number of menaquinone-2 (2-methyl-3-geranyl-1,4-naphthoquinone) derivatives. The 2-des-methyl and 2-ethyl-MK-2 derivatives had very low activity as substrates. The 6- or 7-methyl-MK-2 derivatives and (6,7)-chloro-MK-2 were relatively high Vmax substrates with Km values increased over that seen for K-2. The 5- or 8-methyl-MK-2 derivatives were low Vmax substrates but also demonstrated low Km values. Although these observations suggested that 5-methyl-MK-2 might be a competitive inhibitor of the carboxylation reaction, it was not an effective inhibitor of either phylloquinone or 6-methyl-MK-2-dependent carboxylation.     

Vitamin K-dependent carboxylation of glutamic acid residues to γ-carboxyglutamic acid in lung microsomes

Vitamin K-dependent carboxylation of glutamic acid residues to γ-carboxyglutamic acid was demonstrated in proteins of lung microsomes. The carboxylation was 12% of that in liver microsomes per milligram of mierosomal protein. Carboxylation was very low with microsomes of untreated rats but increased with time up to 42 h after warfarin administration. Carboxylation was highest with microsomes from rats fed a vitamin K-deficient diet. This suggests that a protein(s) accumulates which can be carboxylated in vitro/J. Lung microsomes also catalyzed the vitamin K-dependent carboxylation of the peptide Phe-Leu-Glu-Glu-Leu. The peptide carboxylase activity was 9% of that obtained with liver microsomes. Vitamin K-dependent protein carboxylation required NADH or dithioerythritol, suggesting that vitamin K had to be reduced to the hydroquinone. Accordingly, vitamin K₁ hydroquinone had carboxylating activity without added reducing agents. Menaquinone-3 was considerably more active than phylloquinone. The temperature optimum for carboxylation was around 27 °C.     

Synthesis of trifluoromethyl analogs of vitamin K as substrates for the liver microsomal vitamin K-dependent carboxylase

The rat liver microsomal vitamin K-dependent carboxylase catalyzes the carboxylation of glutamyl to gamma-carboxyglutamyl residues in the presence of reduced vitamin K, O2 and CO2. The specificity of the enzyme for the vitamin substrate has been probed by the synthesis of the trifluoromethyl analogs of menaquinone-2 (2-methyl-3-geranyl-1,4-naphthoquinone) and phylloquinone (2-methyl-3-phytyl-1,4-naphthoquinone). The reduced (naphthohydroquinone) forms of the trifluoromethyl analogs of the natural vitamins had no substrate activity but were competitive inhibitors of the reaction with a Ki in the same range as the Km of the normal substrate. The oxidized form of the trifluoromethyl analogs of vitamin K also caused inhibition by a mechanism that could not be established. Under the incubation conditions utilized, fluorine was lost from the trifluoromethyl group by a process that was dithiothreitol and high pH dependent.     

Vitamin K-dependent carboxylase: Synthesis of an inhibitor of the glutamyl binding site

Liver microsomes contain a vitamin K and O₂-dependent carboxylase that converts peptide-bound glutamyl residues to γ-carboxyglutamyl residues. The peptide Boc-O-phospho—Ser-O-phospho—Ser—Leu-OMe has now been synthesized. This peptide inhibits the carboxylation of endogenous protein precursors by a detergent-solubilized preparation of the carboxylase and is an apparent competitive inhibitor of the carboxylation of Phe—Leu—Glu—Glu—Leu.     

Vitamin K-dependent carboxylase: Evidence for a semiquinone radical intermediate

Nanosecond laser flash photolysis has been used to produce and identify the vitamin K semiquinone (radical) from vitamin K dihydroquinone and to observe its formation and decay in the presence of vitamin K-dependent carboxylase (epoxidase). The activity of vitamin K-dependent carboxylase is not decreased by exposure to the laser. Absorbance of the semiquinone is proportional to enzyme concentration and is stimulated by a synthetic substrate, PheLeuGluGluIle. Stabilization of the semiquinone is observed in the presence of the enzyme. The semiquinone is rapidly destroyed in the presence of inhibitors of vitamin K-dependent carboxylase and vitamin K epoxidase.     

Synthesis of clotting factors by a cell-free system from rat liver in response to the addition of vitamin K1 in vitro.

A cell free system from the liver of vitamin K-deficient rats will form clotting factors after addition of vitamin K₁ in vitro. The response requires both microsomal pellet and supernatant. It is not energy dependent and no co-factor requirement could be demonstrated. Immunological tests and the response to vitamin K₁ analogues demonstrate the physiological nature of the response. It has been recently claimed that vitamin K is required for the formation of calcium binding sites by carboxylation of glutamyl residues. Failure to demonstrate an energy requirement in this system suggests that either vitamin K-dependent carboxylation proceeds by a mechanism hitherto unknown in biology or that the vitamin K-dependent reaction is not directly coupled to carboxylation.     

Characteristics of the vitamin K-dependent carboxylating system in human placenta.

Gamma-carboxyglutamic acid, formed during the post-translational vitamin K-dependent carboxylation of glutamic acid residues in polypeptides has been identified not only in coagulation factors II (prothrombin),, VII, IX and X [1--4], but also in several other plasma proteins [3,5,6] and in protein of bone [7,8] and kidney [9]. In rat liver, carboxylation is mediated through an enzyme system located in the microsomal membrane [10]. The enzyme system requires CO2, O2 and the reduced (hydroquinone) form of the vitamin, as well as a suitable substrate [10,11]. Rat liver microsomes also convert vitamin K1 (phylloquinone) to its stable 2,3-epoxide [12]. Several studies suggest a link between carboxylation and the formation of the epoxide [12--14]. In one of these [14], a survey of rat tissues for vitamin K1 epoxidation revealed that, in addition to liver, this activity was also possessed by kidney, bone, spleen and placenta. In preliminary experiments, vitamin K-dependent carboxylating systems have been found in rat and chick kidney [9], in chick bone [15] and in rat spleen and placenta (unpublished observations). In this communication, we describe some of the basic characteristics of the vitamin K-dependent carboxylating system as found in human placental microsomes.     

Vitamin K-dependent carboxylase: requirements for carboxylation of soluble peptide and substrate specificity.

Rat liver microsomes contain a triton X-100 solubilizable vitamin K-dependent carboxylase activity that converts specific glutamyl residues of precursor proteins to γ-carboxyglutamyl residues. This activity has been studied utilizing synthetic peptides as substrates for the enzyme. When compared to the carboxylation of the endogenous microsomal precursors, the peptide carboxylase activity is more sensitive to the action of various inhibitors, and requires a higher concentration of vitamin K for maximal activity. The apparent K_m for the peptide Phe-Leu-Glu-Glu-Leu was found to be 4 mM. Substrate specificity depends on residues adjacent to the carboxylated Glu residues and macromolecular recognition sites.     

Rabbit liver fructose 1,6-bisphosphatase: labeling of the active and allosteric sites with pyridoxal 5-phosphate and sequence of a nonapeptide from the active site

The active and allosteric sites of fructose 1,6-bisphosphatase (Fru-P₂ase, EC 3.1.3.11) were labeled by reaction with pyridoxal phosphate and sodium borohydride in the presence of the allosteric inhibitor AMP or the substrate, Fru-P₂, respectively. Modification of the active site results in loss of activity. Modification of the allosteric site decreases the sensitivity of the enzyme to inhibition by AMP and alters its ability to bind to blue dextran-Sepharose. The allosteric and active sites have been located on different cyanogen bromide peptides; the sequence of a nonapeptide from the active site is (H)GlyLysLeuArgLeuLeu TyrGluCys(OH). The lysyl residue is modified by pyridoxal phosphate.     

Some characteristics of a vitamin K-dependent carboxylating system from rat liver microsomes

Vitamin K-dependent carboxylation of protein has been obtained in microsomes from K-deficient rats by supplementing the microsomes with either a reduced pyridine nucleotide or dithiothreitol. γ-Carboxyglutamate residues have been directly identified as the radioactive reaction product after incubating the microsomes with NaH¹⁴CO₃ and vitamin K. Evidence is presented that vitamin K is reduced to the hydroquinone prior to its involvement in carboxylation and that dithiothreitol has an additional role of protecting a critical sulfhydryl group. A structure-activity study using a variety of vitamins K and related compounds is reported.     

Intestinal Scavenger Receptors Are Involved in Vitamin K1 Absorption

Vitamin K₁ (phylloquinone) intestinal absorption is thought to be mediated by a carrier protein that still remains to be identified. Apical transport of vitamin K₁ was examined using Caco-2 TC-7 cell monolayers as a model of human intestinal epithelium and in transfected HEK cells. Phylloquinone uptake was then measured ex vivo using mouse intestinal explants. Finally, vitamin K₁ absorption was compared between wild-type mice and mice overexpressing scavenger receptor class B type I (SR-BI) in the intestine and mice deficient in cluster determinant 36 (CD36). Phylloquinone uptake by Caco-2 cells was saturable and was significantly impaired by co-incubation with α-tocopherol (and vice versa). Anti-human SR-BI antibodies and BLT1 (a chemical inhibitor of lipid transport via SR-BI) blocked up to 85% of vitamin K₁ uptake. BLT1 also decreased phylloquinone apical efflux by ∼80%. Transfection of HEK cells with SR-BI and CD36 significantly enhanced vitamin K₁ uptake, which was subsequently decreased by the addition of BLT1 or sulfo-N-succinimidyl oleate (CD36 inhibitor), respectively. Similar results were obtained in mouse intestinal explants. In vivo, the phylloquinone postprandial response was significantly higher, and the proximal intestine mucosa phylloquinone content 4 h after gavage was increased in mice overexpressing SR-BI compared with controls. Phylloquinone postprandial response was also significantly increased in CD36-deficient mice compared with wild-type mice, but their vitamin K₁ intestinal content remained unchanged. Overall, the present data demonstrate for the first time that intestinal scavenger receptors participate in the absorption of dietary phylloquinone.     

Expression and characterization of the naturally occurring mutation L394R in human gamma-glutamyl carboxylase

Patients with mutation L394R in gamma-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 wild-type counterpart, the L394R mutant shows an augmented activation of FLEEL carboxylation by the propeptide.     

Kinetics of carboxylation of endogenous and exogenous substrates by the vitamin K-dependent carboxylase

The vitamin K-dependent carboxylation of the exogenous pentapeptide, Phe-Leu-Glu-Glu-Ile, and endogenous liver microsomal protein was studied in solubilized rat liver microsomes. The MnCl2 stimulation of the vitamin K-dependent pentapeptide carboxylation rate, which is conducted at subsaturating concentrations of pentapeptide, is due to the cation's ability to lower the Km of the substrate. Although there are clear kinetic differences observed between the carboxylation rates for the pentapeptide and the endogenous protein substrates, several lines of evidence suggest that the same carboxylase system is responsible for both. These points of evidence are (i) the initial velocity of endogenous protein carboxylation is lowered in the presence of 3 mM pentapeptide; (ii) the presence of endogenous microsomal protein substrate causes an initial lag in pentapeptide carboxylation; and (iii) this initial lag phase is not observed when the total endogenous substrate pool is carboxylated by a preincubation reaction prior to the addition of pentapeptide.     

The primary structure of the COOH-terminal half of cholera toxin subunit A1 containing the ADP-ribosylation site

The sequence of 96 amino acid residues from the COOH-terminus of the active subunit of cholera toxin, A₁, has been determined as PheAsnValAsnAspVal LeuGlyAlaTyrAlaProHisProAsxGluGlu GluValSerAlaLeuGlyGly IleProTyrSerGluIleTyrGlyTrpTyrArg ValHisPheGlyValLeuAsp GluGluLeuHisArgGlyTyrArgAspArgTyr TyrSerAsnLeuAspIleAla ProAlaAlaAspGlyTyrGlyLeuAlaGlyPhe ProProGluHisArgAlaTrp ArgGluGluProTrpIleHisHisAlaPro ProGlyCysGlyAsnAlaProArg(OH). This is the largest fragment obtained by BrCN cleavage of the subunit A₁ (M_r 23,000), and has previously been indicated to contain the active site for the adenylate cyclase-stimulating activity. Unequivocal identification of the COOH-terminal structure was achieved by separation and analysis of the terminal peptide after the specific chemical cleavage at the only cysteine residue in A₁ polypeptide. The site of self ADP-ribosylation in the A₁ subunit [C. Y. Lai, Q.-C. Xia, and P. T. Salotra (1983) Biochem. Biophys. Res. Commun.116, 341–348] has now been identified as Arg-50 of this peptide, 46 residues removed from the COOH-terminus. The cysteine that forms disulfide bridge to A₂ subunit in the holotoxin is at position 91.     

Regulation of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase in vivo: Control by High CO2 Concentration

High CO₂ concentrations (HC) in air induce partial deactivation of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO, EC 4.1.1.39). Under saturating irradiance, increase in [CO₂] to 1 200 cm³ m^–3 reduces the concentration of operating carboxylation centres by 20–30 %. At a further increase in [CO₂], the activity remained on the same level. Under limiting irradiance, the lowest activity was reached at 600 cm³(CO₂) m^–3. The presence of oxygen diminished deactivation, but O₂ failed to stimulate reactivation under high CO₂. Conditions that favour oxygenation of ribulose-1,5-bisphosphate (RuBP) facilitated reactivation. Even HC did not act as an inhibitor. HC induces deactivation of RuBPCO by increasing the concentration of free reaction centres devoid of the substrate, which are more vulnerable to inhibition than the centres filled with substrates or products.     

Vitamin K-dependent carboxylase: Possible artifact of analysis due to a pyridine nucleotide-dependent carboxylation

Addition of pyridine nucleotides to a microsomal system which is commonly used to study the vitamin K-dependent microsomal carboxylase promoted carboxylation of unknown endogenous compounds. Upon gel filtration, the carboxylated products were found to be of lower molecular weight (MW range 180–650) than the peptide substrate of the vitamin K-dependent carboxylase. Synthesis of these products was not inhibited by vitamin K antagonists nor did pyridine nucleotides stimulate carboxylation of the peptide substrate for vitamin K-dependent carboxylation in the absence of vitamin K. Thus the reaction appears to be mediated by a different enzyme. Dialysis of the microsomal system removed this pyridine nucleotide-stimulated carboxylation and activated the vitamin K-dependent carboxylation and epoxidation reactions. These data point out a possible artifact in the routine study of this enzyme and suggest that dialysis should be carried out prior to studying these two vitamin K-dependent reactions.     

The vitamin K-dependent carboxylation system in human osteosarcoma U2-OS cells. Antidotal effect of vitamin K1 and a novel mechanism for the action of warfarin.

An osteoblast-like human osteosarcoma cell line (U2-OS) has been shown to possess a vitamin K-dependent carboxylation system which is similar to the system in human HepG2 cells and in liver and lung from the rat. In an 'in vitro' system prepared from these cells, vitamin K1 was shown to overcome warfarin inhibition of gamma-carboxylation carried out by the vitamin K-dependent carboxylase. The data suggest that osteoblasts, the cells involved in synthesis of vitamin K-dependent proteins in bone, can use vitamin K1 as an antidote to warfarin poisoning if enough vitamin K1 can accumulate in the tissue. Five precursors of vitamin K-dependent proteins were identified in osteosarcoma and HepG2 cells respectively. In microsomes (microsomal fractions) from the osteosarcoma cells these precursors revealed apparent molecular masses of 85, 78, 56, 35 and 31 kDa. When osteosarcoma cells were cultured in the presence of warfarin, vitamin K-dependent 14C-labelling of the 78 kDa precursor was enhanced. Selective 14C-labelling of one precursor was also demonstrated in microsomes from HepG2 cells and from rat lung after warfarin treatment. In HepG2 cells this precursor was identified as the precursor of (clotting) Factor X. This unique 14C-labelling pattern of precursors of vitamin K-dependent proteins in microsomes from different cells and tissues reflects a new mechanism underlying the action of warfarin.     

Vitamin K-dependent gamma-glutamyl carboxylase activity in the chick embryonic chorioallantoic membrane.

During embryonic development of the chick, the onset of calcium transport by the chorioallantoic membrane (CAM) is concomitant with the appearance of a calcium-binding protein (CaBP). The development-specific expression of the CaBP in the CAM is inhibited by vitamin K antagonism in ovo with the anticoagulant, warfarin. However, the CaBP remains immunologically detectable in the CAM of warfarin-treated embryos, suggesting the presence of a precursor form of the CaBP. Previously, we have demonstrated that CaBP expression in CAM organ cultures is inducible by vitamin K. Furthermore, the CaBP contains several residues of the modified amino acid, gamma-carboxyglutamic acid (gamma-CGlu), which has been shown to be formed by vitamin K-dependent carboxylation of glutamic acid in several plasma clotting proteins. This study reports the presence of a post-translational, vitamin K-dependent gamma-glutamyl carboxylase activity in the CAM. Our results show that explants of CAM incorporate H14CO3 in an age-specific and vitamin K-dependent manner. Incorporation of H14CO3 by the CAM is further potentiated by warfarin treatment of the embryos, presumably owing to an elevation of the amount of endogenous uncarboxylated protein precursor(s). Among the subcellular (nuclear, mitochondrial, microsomal, and soluble) fractions of the CAM, only microsomes exhibit specific incorporation of of H14CO3 into gamma-CGlu. The CAM microsomal carboxylation activity is post-translational, vitamin K-dependent, specific for prenylated homologs of vitamin K, sensitive to warfarin, and appears to be unrelated to the activities of biotin-dependent carboxylases or phosphoenolpyruvate carboxykinase. Optimal carboxylation activity occurs after incubation of the microsomes with H14CO3 for 60 min at 37 degrees C in the presence of over 100 microgram of vitamin K1/ml.     

Carboxylation, the completion step in prothrombin biosynthesis

It has been found that [¹⁴C]CO₂ is incorporated into prothrombin $\text{in vivo}$ in two hours. The amount of incorporation is increased 3 to 4 fold by the administration of vitamin K₁ to the warfarin-treated vitamin K-deficient rat, over incorporation in the “normal” rat. The radioactivity is found in one acidic peptide following trypsin digestion and following pronase and aminopeptidase digestion is found in one acidic amino acid. The [¹⁴C] is lost on heating of this amino acid at pH 2, leaving unlabeled glutamic acid. It appears that the vitamin K-dependent step in the “completion” of prothrombin is carboxylation of a glutamyl residue of the preformed protein molecule.