Relationship of dithiothreitol-dependent microsomal vitamin K quinone and vitamin K epoxide reductases inhibition of epoxide reduction by vitamin K quinone

Vitamin K quinone was shown to be an effective inhibitor of vitamin K epoxide reduction by whole rat liver microsomes. Observation of inhibition was dependent upon the mode of addition of the substrate and inhibitor suggesting segregation of the compounds into different microsomal vesicles under certain conditions. The result is consistent with reduction of both vitamin K quinone and vitamin K epoxide by a single enzyme or a multisite enzyme complex.     

Vitamin K, 2,3-Epoxide And Quinone Reduction: Mechanism And Inhibition

The chemical and enzymatic pathways of vitamin K₁ epoxide and quinone reduction have been investigated. The reduction of the epoxide by thiols is known to involve a thiol-adduct and a hydroxy vitamin K enolate intermediate which eliminates water to yield the quinone. Sodium borohydride treatment resulted in carbonyl reduction generating relatively stable compounds that did not proceed to quinone in the presence of base. NAD(P)H:quinone oxidoreductase (DT-diaphorase. E.C. I.6.99.2) reduction of vitamin K to the hydroquinone was a significant process in intact microsomes. but 1/5th the rate of the dithiothreitol (DTT)-dependent reduction. No evidence was found for DT-diaphorase catalyzed reduction of vitamin K₁ epoxide, nor was it capable of mediating transfer of electrons from NADH to the microsomal epoxide reducing enzyme. Purified diaphorase reduced detergent- solubilized vitamin K, 10^?5 as rapidly as it reduced dichlorophenylindophenol(DCPIP). Reduction of 10 μM vitamin K, by200 μM NADH was not inhibited by 10μM dicoumarol. whereas DCPIP reduction was fully inhibited. In contrast to vitamin K, (menadione). vitamin K₁ (phylloquinone) did not stimulate microsomal NADPH consumption in the presence or absence of dicoumarol. DTT-dependent vitamin K epoxide reduction and vitamin K reduction were shown to be mutually inhibitory reactions. suggesting that both occur at the same enzymatic site. On this basis, a mechanism for reduction of the quinone by thiols is proposed. Both the DTT-dependent reduction of vitamin K₁ epoxide and quinone. and the reduction of DCPIP by purified DT-diaphorase were inhibited by dicoumarol, warfarin. lapachol. and sulphaquinoxaline     

Lapachol inhibition of vitamin K epoxide reductase and vitamin K quinone reductase

Lapachol [2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone] has been shown to be a potent inhibitor of both vitamin K epoxide reductase and the dithiothreitol-dependent vitamin K quinone reductase of rat liver microsomes in vitro. These observations explain the anticoagulant activity of lapachol previously observed in both rats and humans. Lapachol inhibition of the vitamin K epoxide and quinone reductases resembled coumarin anticoagulant inhibition, and was observed in normal strain but not in warfarin-resistant strain rat liver microsomes. This similarity of action suggests that the lactone functionality of the coumarins is not critical for their activity. The initial-velocity steady-state inhibition patterns for lapachol inhibition of the solubilized vitamin K epoxide reductase were consistent with tight binding of lapachol to the oxidized form of the enzyme, and somewhat lower affinity for the reduced form. It is proposed that lapachol assumes a 4-enol tautomeric structure similar to that of the 4-hydroxy coumarins. These structures are analogs of the postulated hydroxyvitamin K enolate intermediate bound to the oxidized form of the enzyme in the chemical reaction mechanism of vitamin K epoxide reductase, thus explaining their high affinity.     

Rat and human liver vitamin K epoxide reductase: inhibition by thiol blockers and vitamin K1

1. Reduction of vitamin K1 2,3-epoxide by rat and human liver vitamin K epoxide reductase is inhibited by N-ethylmaleimide and iodoacetamide. 2. Both enzymes are protected from inhibition by N-ethylmaleimide by vitamin K1 or vitamin K1 2,3-epoxide. 3. Vitamin K1 inhibits reduction of vitamin K1 2,3-epoxide to vitamin K1 which suggests product inhibition of the enzyme.     

Vitamin K epoxide reductase: homology, active site and catalytic mechanism

Vitamin K epoxide reductase (VKOR) recycles reduced vitamin K, which is used subsequently as a co-factor in the gamma-carboxylation of glutamic acid residues in blood coagulation enzymes. VKORC1, a subunit of the VKOR complex, has recently been shown to possess this activity. Here, we show that VKORC1 is a member of a large family of predicted enzymes that are present in vertebrates, Drosophila, plants, bacteria and archaea. Four cysteine residues and one residue, which is either serine or threonine, are identified as likely active-site residues. In some plant and bacterial homologues the VKORC1 homologous domain is fused with domains of the thioredoxin family of oxidoreductases. These might reduce disulfide bonds of VKORC1-like enzymes as a prerequisite for their catalytic activities.     

Vitamin K dependent carboxylase: subcellular location of the carboxylase and enzymes involved in vitamin K metabolism in rat liver

Vitamin K dependent carboxylation of an exogenous peptide substrate and endogenous protein substrates, vitamin K epoxidation, and reduction of vitamin K epoxide were measured in subcellular fractions from rat liver. The rough microsomal fraction was highly enriched in all four activities; lower levels were found in smooth microsomes. Mitochondria, nuclei, and cytosol had negligible activities. The addition of 0.2% Triton X-100 to intact microsomes resulted in a 10-20-fold stimulation in carboxylation of a peptide substrate. This marked latency suggests that the active site of the carboxylase may be accessible only from the lumen of the microsomal membrane. A lumen-facing orientation of the carboxylase was also supported by its inaccessibility to trypsin in intact microsomes contrasted with marked inhibition by trypsin in detergent-permeabilized microsomes. Vitamin K epoxidase and epoxide reductase activities were also inhibited by trypsin much more effectively in permeabilized than in intact microsomes, although some degree of exposure at the cytosolic surface was also indicated. These data suggest that carboxylation is an early event in prothrombin synthesis occurring primarily on the lumen side of the rough endoplasmic reticulum membrane. The location of the vitamin K epoxidation-reduction cycle enzymes is consistent with their possible role in the carboxylation reaction.     

Formation of hydroxyvitamin K by vitamin K epoxide reductase of warfarin-resistant rats

A new metabolite of vitamin K, 2(3)-hydroxy-2,3-dihydro-2-methyl,3-phytyl-1,4-naphthoquinone (hydroxyvitamin K), has been identified as a product of vitamin K epoxide metabolism in hepatic microsomes from warfarin-resistant rats, but not in those derived from normal rats. The structure was determined by comparison of the high performance liquid chromatography retention times, UV, IR, CD, and mass spectra of the unknown with chemically synthesized standards. Alterations in the formation of hydroxyvitamin K occur in parallel with alterations in total vitamin K epoxide conversion with respect to reaction time, extent of reaction, detergent stimulation, and inhibition by warfarin. Thus, hydroxyvitamin K appears to be a product of the warfarin-resistant vitamin K epoxide reductase. It is neither a substrate nor an inhibitor of epoxide reduction. Hydroxyvitamin K is formed from both enantiomers of racemic vitamin K epoxide with little stereoselectivity for the configuration of either the oxirane ring or the phytyl side chain. The reaction is stereospecific; however, the biologically formed (+)-vitamin K epoxide yields exclusively (+)-3-hydroxyvitamin K. Observation of this product is discussed as a key to understanding the normal reaction mechanism of the enzyme.     

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.     

Vitamin k epoxide reductase: a protein involved in angiogenesis

Vitamin K epoxide reductase (VKOR) is a newly identified protein which has been reported to convert the epoxide of vitamin K back to vitamin K, a cofactor essential for the posttranslational gamma-carboxylation of several blood coagulation factors. We found that the gene is expressed ubiquitously including vascular endothelial cells, smooth muscle cells, fibroblasts and cardiomyocytes, and is overexpressed in 11 tumor tissues on microarray. Stable transfection of VKOR cDNA into tumor cell line A549 and H7402 did not promote the cell proliferation. These results promoted us to hypothesize that VKOR may also be involved in angiogenesis. To test this hypothesis, the expression of VKOR was studied in different vascular cells in developmental and pathologic heart tissues. The effects of overexpression and suppressing expression of VKOR on endothelial cell proliferation, migration, adhesion, and tubular network formation were explored. We found that VKOR expression in arteries was prominent in vascular endothelial cells and was high in the ventricular aneurysm tissue of human heart and human fetal heart. In vitro studies showed that overexpression of VKOR slightly but significantly stimulated human umbilical vein endothelial cell proliferation (by 120%), migration (by 118%), adhesion (by 117%), as well as tubular network formation. Antisense to VKOR gene inhibited the proliferation (by 67%), migration (by 64%), adhesion (by 50%), and tubular network formation. Our findings support the impact of VKOR in the process of angiogenesis; hence, the molecule may have a potential application in cardiovascular disease and cancer therapy.     

Solubilization and characterization of vitamin K epoxide reductase from normal and warfarin-resistant rat liver microsomes

Two procedures have been developed for the solubilization of vitamin K epoxide reductase from rat liver microsomal membranes using the detergent Deriphat 160 at pH 10.8. The methods are applicable to both normal and Warfarin-resistant-strain rat liver microsomes and yield material suitable for further purification. The preparations retain dithiothreitol-dependent vitamin K quinone reductase activity as well as vitamin K epoxide reductase and are free of vitamin K-dependent carboxylase and epoxidase activities. Optimal epoxide reductase activity is obtained at 0.1 M KCl and pH 9 in the presence of sodium cholate. Artifactual formation of vitamin K metabolites was eliminated through the use of mercuric chloride to remove excess dithiothreitol prior to extraction and metabolite assay. Using the solubilized enzyme, valid initial velocities were measured, and reproducible kinetic data was obtained. The substrate initial velocity patterns were determined and are consistent with a ping-pong kinetic mechanism. The kinetic parameters obtained are a function of the cholate concentration, but do not vary drastically from those obtained using intact microsomal membranes. At 0.8% cholate, the enzymes solubilized from normal Warfarin-sensitive- and Warfarin-resistant-strain rat livers exhibit respective values of Vmax = 3 and 0.75 nmol/min/g liver; Km for vitamin K epoxide = 9 and 4 microM; and Km for dithiothreitol of 0.6 and 0.16 mM.     

Anti-oxidant/pro-oxidant reactions of vitamin K

Experiments were designed to measure O2 consumption caused by the oxidation of linoleic acid. These experiments show that vitamin K has antioxidant activity and that the reduction in linoleic acid oxidation is directly dependent upon vitamin K concentration. Conversely, vitamin K hydroquinone enhances linoleic acid oxidation in the absence of iron catalyst, again in a concentration dependent manner. At equilmolar concentrations vitamin K is about 80% as effective as vitamin E as an antioxidant. Vitamin E inhibits the oxidation of linoleic acid catalyzed by vitamin K hydroquinone. Vitamin E also strongly inhibits vitamin K dependent formation of both vitamin K epoxide and gamma-carboxyglutamic acid (gla). The significance of these observations to vitamin K action in vivo is discussed.     

The conversion of vitamin K epoxide to vitamin K quinone and vitamin K quinone to vitamin K hydroquinone uses the same active site cysteines

Vitamin K epoxide (or oxido) reductase (VKOR) is the target of warfarin and provides vitamin K hydroquinone for the carboxylation of select glutamic acid residues of the vitamin K-dependent proteins which are important for coagulation, signaling, and bone metabolism. It has been known for at least 20 years that cysteines are required for VKOR function. To investigate their importance, we mutated each of the seven cysteines in VKOR. In addition, we made VKOR with both C43 and C51 mutated to alanine (C43A/C51A), as well as a VKOR with residues C43-C51 deleted. Each mutated enzyme was purified and characterized. We report here that C132 and C135 of the CXXC motif are essential for both the conversion of vitamin K epoxide to vitamin K and the conversion of vitamin K to vitamin K hydroquinone. Surprisingly, conserved cysteines, 43 and 51, appear not to be important for either reaction. For the in vitro reaction driven by dithiothreitol, the 43-51 deletion mutation retained 85% and C43A/C51A 112% of the wild-type activity. The facile purification of the nine different mutations reported here illustrates the ease and reproducibility of VKOR purification by the method reported in our recent publication [Chu, P.-H., Huang, T.-Y., Williams, J., and Stafford, D. W. (2006) Proc. Natl. Acad. Sci. U S A. 103, 19308-19313].     

A hetero-dimer model for concerted action of vitamin K carboxylase and vitamin K reductase in vitamin K cycle

Vitamin K carboxylase (VKC) is believed to convert vitamin K, in the vitamin K cycle, to an alkoxide-epoxide form which then reacts with CO₂ and glutamate to generate γ-carboxyglutamic acid (Gla). Subsequently, vitamin K epoxide reductase (VKOR) is thought to convert the alkoxide-epoxide to a hydroquinone form. By recycling vitamin K, the two integral-membrane proteins, VKC and VKOR, maintain vitamin K levels and sustain the blood coagulation cascade. Unfortunately, NMR or X-ray crystal structures of the two proteins have not been characterized. Thus, our understanding of the vitamin K cycle is only partial at the molecular level. In this study, based on prior biochemical experiments on VKC and VKOR, we propose a hetero-dimeric form of VKC and VKOR that may explain the efficient oxidation and reduction of vitamin K during the vitamin K cycle.     

Absorption and metabolism of vitamin K in Swiss 3T3 mouse fibroblasts--a model system for study of vitamin K absorption and metabolism

The vitamin K cycle previously described in liver has been demonstrated in Swiss 3T3 mouse fibroblasts. Vitamin K epoxide and gamma-carboxyglutamic acid were isolated from the cells and chemically characterized. Menaquinone (MK4) is also metabolized to its epoxide and vitamin K epoxide is reduced to vitamin K in these cells. Thus Swiss 3T3 mouse fibroblasts provide a useful model system for the study of vitamin K metabolism. Possible functions of the vitamin K-dependent protein(s) in fibroblasts are discussed.     

Membrane topology mapping of vitamin K epoxide reductase by in vitro translation/cotranslocation

Vitamin K epoxide reductase (VKOR) catalyzes the conversion of vitamin K 2,3-epoxide into vitamin K in the vitamin K redox cycle. Recently, the gene encoding the catalytic subunit of VKOR was identified as a 163-amino acid integral membrane protein. In this study we report the experimentally derived membrane topology of VKOR. Our results show that four hydrophobic regions predicted as the potential transmembrane domains in VKOR can individually insert across the endoplasmic reticulum membrane in vitro. However, in the intact enzyme there are only three transmembrane domains, residues 10-29, 101-123, and 127-149, and membrane-integration of residues 75-97 appears to be suppressed by the surrounding sequence. Results of N-linked glycosylation-tagged full-length VKOR shows that the N terminus of VKOR is located in the endoplasmic reticulum lumen, and the C terminus is located in the cytoplasm. Further evidence for this topological model of VKOR was obtained with freshly prepared intact microsomes from insect cells expressing HPC4-tagged full-length VKOR. In these experiments an HPC4 tag at the N terminus was protected from proteinase K digestion, whereas an HPC4 tag at the C terminus was susceptible. Altogether, our results suggest that VKOR is a type III membrane protein with three transmembrane domains, which agrees well with the prediction by the topology prediction program TMHMM.     

Thematic Review Series: Fat-Soluble Vitamins: Vitamin K: Recent trends in the metabolism and cell biology of vitamin K with special reference to vitamin K cycling and MK-4 biosynthesis

In contrast to other fat-soluble vitamins, dietary vitamin K is rapidly lost to the body resulting in comparatively low tissue stores. Deficiency is kept at bay by the ubiquity of vitamin K in the diet, synthesis by gut microflora in some species, and relatively low vitamin K cofactor requirements for γ-glutamyl carboxylation. However, as shown by fatal neonatal bleeding in mice that lack vitamin K epoxide reductase (VKOR), the low requirements are dependent on the ability of animals to regenerate vitamin K from its epoxide metabolite via the vitamin K cycle. The identification of the genes encoding VKOR and its paralog VKOR-like 1 (VKORL1) has accelerated understanding of the enzymology of this salvage pathway. In parallel, a novel human enzyme that participates in the cellular conversion of phylloquinone to menaquinone (MK)-4 was identified as UbiA prenyltransferase-containing domain 1 (UBIAD1). Recent studies suggest that side-chain cleavage of oral phylloquinone occurs in the intestine, and that menadione is a circulating precursor of tissue MK-4. The mechanisms and functions of vitamin K recycling and MK-4 synthesis have dominated advances made in vitamin K biochemistry over the last five years and, after a brief overview of general metabolism, are the main focuses of this review.     

Determination of plasma Vitamin K by high-performance liquid chromatography with fluorescence detection using Vitamin K analogs as internal standards

A HPLC fluorescence determination method for Vitamin K derivatives (Vitamin K(1), phylloquinone, PK and K(2), menaquinones, MK-4 and MK-7) using post-column reduction and internal standards was developed. Selectivity and reproducibility were increased by optimized chromatography conditions and satisfactory precision and accuracy were attained by using synthetic internal standards. After addition of internal standards to plasma samples, lipids were extracted with ethanol and hexane. Chromatography was performed by isocratic reverse phase separation on a C18 column. Vitamin K derivatives were detected at 430 nm with excitation at 320 nm for MK-4 and 240 nm for PK and MK-7. The detection limits for MK-4, PK and MK-7 were 4, 2 and 4 pg, respectively. The recoveries of MK-4, PK and MK-7 were greater than 92% and the inter- and intra-assay R.S.D. values were 5.7-9.2% for MK-4, 4.9-9.6% for PK and 6.3-19.3% for MK-7. The data showed good correlation between proposed method and LC-APCI/MS method for MK-4 (R(2)=0.988), PK (R(2)=0.979) and MK-7 (R(2)=0.986). The method allows the determination of Vitamin K for evaluating their clinical and nutritional status.     

Conserved Loop Cysteines of Vitamin K Epoxide Reductase Complex Subunit 1-like 1 (VKORC1L1) Are Involved in Its Active Site Regeneration

Vitamin K epoxide reductase complex subunit 1 (VKORC1) reduces vitamin K epoxide in the vitamin K cycle for post-translational modification of proteins that are involved in a variety of biological functions. However, the physiological function of VKORC1-like 1 (VKORC1L1), a paralogous enzyme sharing about 50% protein identity with VKORC1, is unknown. Here we determined the structural and functional differences of these two enzymes using fluorescence protease protection (FPP) assay and an in vivo cell-based activity assay. We show that in vivo VKORC1L1 reduces vitamin K epoxide to support vitamin K-dependent carboxylation as efficiently as does VKORC1. However, FPP assays show that unlike VKORC1, VKORC1L1 is a four-transmembrane domain protein with both its termini located in the cytoplasm. Moreover, the conserved loop cysteines, which are not required for VKORC1 activity, are essential for VKORC1L1''s active site regeneration. Results from domain exchanges between VKORC1L1 and VKORC1 suggest that it is VKORC1L1''s overall structure that uniquely allows for active site regeneration by the conserved loop cysteines. Intermediate disulfide trapping results confirmed an intra-molecular electron transfer pathway for VKORC1L1''s active site reduction. Our results allow us to propose a concerted action of the four conserved cysteines of VKORC1L1 for active site regeneration; the second loop cysteine, Cys-58, attacks the active site disulfide, forming an intermediate disulfide with Cys-139; the first loop cysteine, Cys-50, attacks the intermediate disulfide resulting in active site reduction. The different membrane topologies and reaction mechanisms between VKORC1L1 and VKORC1 suggest that these two proteins might have different physiological functions.     

A comparison of warfarin resistance and liver microsomal vitamin K epoxide reductase activity in rats

Vitamin K-1 epoxide reductase activity was investigated in liver microsomal preparations from warfarin-resistant and -susceptible rats. One rat strain (TAS) is susceptible to the anticoagulant and lethal effects of warfarin and the other two strains are homozygous for warfarin resistance genes from either wild Welsh (HW) or Scottish (HS) rats. The enzyme in microsomal preparations from HW rat livers apparently has a reduced affinity for both warfarin and vitamin K-1 2,3-epoxide. The kinetic parameters for the enzyme activity in HS microsomal preparations indicated, however, that vitamin K-1 epoxide reductase in this warfarin-resistant strain was very similar, in respect of substrate and inhibitor affinities, to that prepared from susceptible (TAS) animals. Analysis of vitamin K-1 epoxide reductase activity in the livers of animals that had been orally treated with sodium warfarin (20 mg/kg body wt.) indicated that enzyme activity was inhibited in all three strains, although this dose is lethal only to animals of the TAS strain.     

Effect of N-ethylmaleimide on beef and rat liver vitamin K1 epoxide reductase

There is little difference in the extent of inactivation of beef liver microsomal vitamin K1 epoxide reductase by N-ethylmaleimide (NEM) whether or not the microsomes are pre-treated with dithiothreitol (DTT). The rat liver microsomal enzyme, however, is inactivated by NEM to a much greater extent if the microsomes are pre-treated with DTT. The beef liver enzyme activity is protected from NEM inactivation by the substrate, vitamin K1 epoxide. Ping-pong kinetics are exhibited by the beef liver enzyme. These results support a mechanism for vitamin K1 epoxide reductase in which the function of the required dithiol is to reduce an active site disulfide bond; however, the geometry of the active sites of the enzyme from rat and beef may be different.