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.     

Human vitamin K 2,3-epoxide reductase complex subunit 1-like 1 (VKORC1L1) mediates vitamin K-dependent intracellular antioxidant function

Human vitamin K 2,3-epoxide reductase complex subunit 1-like 1 (VKORC1L1), expressed in HEK 293T cells and localized exclusively to membranes of the endoplasmic reticulum, was found to support both vitamin K 2,3-epoxide reductase (VKOR) and vitamin K reductase enzymatic activities. Michaelis-Menten kinetic parameters for dithiothreitol-driven VKOR activity were: K(m) (μM) = 4.15 (vitamin K(1) epoxide) and 11.24 (vitamin K(2) epoxide); V(max) (nmol·mg(-1)·hr(-1)) = 2.57 (vitamin K(1) epoxide) and 13.46 (vitamin K(2) epoxide). Oxidative stress induced by H(2)O(2) applied to cultured cells up-regulated VKORC1L1 expression and VKOR activity. Cell viability under conditions of no induced oxidative stress was increased by the presence of vitamins K(1) and K(2) but not ubinquinone-10 and was specifically dependent on VKORC1L1 expression. Intracellular reactive oxygen species levels in cells treated with 2,3-dimethoxy-1,4-naphthoquinone were mitigated in a VKORC1L1 expression-dependent manner. Intracellular oxidative damage to membrane intrinsic proteins was inversely dependent on VKORC1L1 expression and the presence of vitamin K(1). Taken together, our results suggest that VKORC1L1 is responsible for driving vitamin K-mediated intracellular antioxidation pathways critical to cell survival.     

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 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].     

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.     

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.     

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.     

Inhibition of the development of metastases by dietary vitamin C:K3 combination

The tumor growth-inhibiting and chemo-potentiating effects of vitamin C and K(3)combinations have been demonstrated both in vitro and in vivo. The purpose of this study was to investigate the influence of orally administered vitamin C and K(3) on the metastasis of mouse liver tumor (T.L.T.) cells implanted in C3H mice. Adult male C3H mice were given water containing vitamin C and K3 (15 g/0.15 g dissolved in 1000 ml) beginning 2 weeks before tumor transplantation until the end of the experiment. T.L.T. cells (106) were implanted intramuscularly in the right thigh of mice. All mice were sacrificed 42 days after tumor transplantation. Primary tumor, lungs, lymph nodes and other organs or tissues suspected of harboring metastases were macroscopically examined. Samples of primary tumors, their local lymph nodes, lungs and main organs such as liver, kidneys, spleen were taken for histological examination. Forty-two percent of control mice exhibited lung metastases and 27% possessed metastases in local lymph nodes whereas 24% of vitamin-treated mice exhibited lung metastases and 10% possessed local lymph nodes metastases. The total number of lung metastases was 19 in control group and 10 in vitamin C and K(3)-treated mice. Histopathological examination of the metastatic tumors from the vitamin-treated mice revealed the presence of many tumor cells undergoing autoschizic cell death. These results demonstrate that oral vitamin C and K(3) significantly inhibited the metastases of T.L.T. tumors in C3H mice. At least a portion of this inhibition was due to tumor cell death by autoschizis.     

Comparison of the vitamins K1, K2 and K3 as cofactors for the hepatic vitamin K-dependent carboxylase

The vitamins phylloquinone (K1), menadione (K3) and various menaquinones (K2) were compared for their ability to serve as a cofactor for the hepatic vitamin K-dependent carboxylase. It was found that the cofactor activity of the menaquinones varied with the length of the aliphatic side-chain and showed an optimum at MK-3. Menadione was not active at all. The concentration required for half-maximal reaction velocity (K 1/2) was determined for the various menaquinones and decreased at increasing chain length. The K 1/2 value for MK-4 was 3-times lower than that for vitamin K1. Under our in vitro conditions both vitamin K1 and the K2 vitamins were rapidly metabolized into a mixture of the quinone, the hydroquinone and the epoxide form. The fact that at equilibrium the level of these three metabolites was independent of the starting material shows that the vitamin K cycle is operational for vitamin K1 as well as for K2.     

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.     

B16 tumor cells contain a warfarin sensitive vitamin K1 2,3 epoxide reductase

Using an adapted assay that requires an enzyme aliquot that forms only 5 pmoles vitamin K, we were able to demonstrate vitamin K1 2,3 epoxide reductase activity in cultured B16 mouse melanoma cells. The enzyme uses dithiothreitol, but not NADH as a reducing cofactor and is sensitive to inhibition by warfarin (2% residual activity at 10 micrograms/ml warfarin). Incubation of B16 cells in culture with 30 micrograms/ml warfarin leads to an 45% residual reductase as compared to normally cultured B16 cells. Combined with the reported presence of vitamin K dependent carboxylase in B16 cells and the cytotoxicity of warfarin towards B16 cells this suggests an active vitamin K cycle in these melanoma cells that may be essential for survival.     

Synthesis of vitamin K1 2,3-epoxide in rat liver mitochondria

Metabolism of vitamin K1 in rat liver mitochondria has been studied with succinate as the source of reducing equivalents. A metabolite was isolated that comigrated with vitamin K1 epoxide using four different chromatographic systems. The purified metabolite had an ultraviolet spectrum (200-330 nm) that was identical to that of synthetic vitamin K1 epoxide. The mass spectrum of the purified metabolite was identical to that of synthetic vitamin K1 epoxide. A comparison of production of vitamin K1 epoxide by mitochondrial and microsomal preparations indicates that the mitochondrial production of vitamin K1 epoxide was about 50% of that of the microsomes. Since the mitochondrial preparation was found to have only 3.4% of the glucose-6-phosphatase activity of the microsomal preparation, it can be concluded that the vitamin K1 epoxide isolated from the mitochondrial incubations was due primarily to mitochondrial synthesis. Epoxidation of vitamin K1 in mitochondria suggests that mitochondria might be sites for vitamin K-dependent carboxylation of protein(s).     

Normal and warfarin-resistant rat hepatocyte metabolism of vitamin K 2,3-epoxide: evidence for multiple pathways of hydroxyvitamin K formation

Vitamin K and 3- (and/or 2)-hydroxy-2,3-dihydro-2-methyl-3-phytyl-1,4-naphthoquinone (hydroxyvitamin K) have been identified as metabolites of vitamin K 2,3-epoxide incubated with hepatocytes isolated from normal and warfarin-resistant rats. Dithiothreitol added to the extracellular medium differentially enhanced the formation of both metabolites: hydroxyvitamin K formation, almost undetectable in the absence of dithiothreitol, was particularly affected. Addition of the vitamin K 2,3-epoxide reductase inhibitors warfarin (5 to 100 microM) and brodifacoum (1 to 5 microM) to normal rat hepatocyte cultures produced a slight increase in hydroxyvitamin K formation and a marked inhibition of vitamin K formation. Brodifacoum was a weak inhibitor of hydroxyvitamin K formation at higher concentrations. Hepatocytes from warfarin-resistant rats catalyzed hydroxyvitamin K formation 1.5 to 2 times faster and vitamin K formation 1.5 to 2 times slower than did normal rat hepatocytes. The addition of warfarin to these cultures had no effect on epoxide metabolism to hydroxyvitamin K and only partially diminished metabolism to vitamin K. In contrast, brodifacoum (1 microM) addition produced 50% inhibition of hydroxyvitamin K formation and almost complete inhibition of vitamin K formation. These data suggest that in resistant, but not in normal rat hepatocytes, the vitamin K 2,3-epoxide reductase makes a significant contribution to hydroxyvitamin K formation. A second sulfhydryl-dependent pathway, present in both strains, is also involved in the formation of this metabolite. They also suggest that in resistant rats, warfarin inhibition of the vitamin K 2,3-epoxide reductase, and presumably the sulfhydryl-dependent vitamin K reductase, is incomplete and independent of concentration.     

Metabolism of vitamin K and prothrombin synthesis: anticoagulants and the vitamin K--epoxide cycle.

Vitamin K is primarily located in hepatic microsomes, where the vitamin K-dependent carboxylation in prothrombin synthesis occurs. Recent evidence supports the idea that the carboxylation is linked to the metabolism of the vitamin--specifically the cyclic interconversion of vitamin K and vitamin K epoxide. The primary site of action of coumarin and indandione anticoagulants appears to be an inhibition of the epoxide-to-vitamin K conversion in this cycle. There is a correlation between the inhibition of prothrombin synthesis and the regeneration of vitamin K from the epoxide by anticoagulants. In hamsters and warfarin-resistant rats prothrombin synthesis and the epoxide-K conversion are less sensitive to warfarin than in the normal rat. The epoxide-K conversion is impaired in resistant rats, which may explain their high vitamin K requirement. There is also a correlation between vitamin K epoxidation and vitamin K-dependent carboxylation, but the apparent link may be because vitamin K hydroquinone is an intermediate in the formation of the epoxide and also the active form in carboxylation. The vitamin K-epoxide cycle is found in extrahepatic tissues such as kidney, spleen, and lung and is inhibited by warfarin.     

Identification of a warfarin-sensitive protein component in a 200S rat liver microsomal fraction catalyzing vitamin K and vitamin K 2,3-epoxide reduction

A partially purified, 200S submicrosomal fraction exhibiting thiol-dependent vitamin K1 (vitamin K) and epoxide reductase activities has been isolated by partial solubilization of rat hepatic microsomes with sodium cholate and separation by centrifugation at 105 000 g into a discontinuous sucrose gradient. At pH 7.4, the rates of vitamin K and vitamin K 2,3-epoxide reduction per milligram of 200S fraction protein were equivalent and were 2.5-3.0 times faster than in microsomes. Reduction of vitamin K 2,3-epoxide occurred in a tightly coupled, two-step reaction initially to vitamin K and subsequently to vitamin K hydroquinone (vitamin KH2). Incorporation of glycerol or sucrose and of sodium cholate into reaction mixtures equivalently affected the rates of both vitamin K and vitamin K 2,3-epoxide reduction, but in the case of epoxide metabolism, the ratios of vitamin KH2/vitamin K were much lower, suggesting that the second reaction has been partially uncoupled from the first. A 14 000-17 000-dalton warfarin-sensitive protein (WSP) that participates in vitamin K and vitamin K 2,3-epoxide reduction in the 200S fraction was identified by incorporation of N-[3H]ethylmaleimide ([3H]NEM) into the catalytically active reduced form of one or more attached disulfides. Reduction of WSP with dithiothreitol was required for reaction with [3H]NEM, and the substrates vitamin K and vitamin K 2,3-epoxide and the inhibitor warfarin all effectively blocked the reaction. 2-Mercaptoethanol could not substitute for dithiothreitol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dioxygen transfer during vitamin K dependent carboxylase catalysis.

The vitamin K dependent carboxylase of liver microsomes is involved in the posttranslational modification of certain serine protease zymogens which are critical components of the blood clotting cascade. During coupled carboxylation/oxygenation this carboxylase converts glutamate residues, dihydrovitamin K, CO2, and O2 to a gamma-carboxyglutamyl (Gla) residue, vitamin K (2R,3S)-epoxide, and H2O with a stoichiometry of 1:1 for all substrates and products. In this paper we investigate the role of molecular oxygen in the reaction by following the course of the oxygen atoms using 18O2. Two different mass spectroscopic techniques, electron ionization positive ion mass spectrometry and supercritical fluid chromatography-negative ion chemical ionization mass spectrometry, were used to quantitate the amount of 18O incorporation into the various oxygens of the vitamin K epoxide product. We found that 0.95 mol atoms of oxygen were incorporated into the epoxide oxygen, 0.05 mol atoms of oxygen were incorporated into the quinone oxygen of vitamin K epoxide, and the remaining ca. 1.0 mol atoms of oxygen were incorporated into H2O. No incorporation of oxygen into vitamin K epoxide from 50% H2(18)O was observed. Thus, the carboxylase operates as a dioxygenase 5% of the time during carboxylation/oxygenation. The relevance of these findings with respect to the nonenzymic "basicity enhancement" model proposed by Ham and Dowd [(1990) J. Am. Chem. Soc. 112, 1660-1661] is discussed.     

Temporal variation in the effects of warfarin on the vitamin K cycle

In this in vivo study, the time-dependent effect of oral sodium warfarin was studied in male rats synchronized under a 12-hr light-dark cycle (light 0600-1800). Groups of 5 animals received an oral dose of 500 micrograms/kg of warfarin or saline at 0600 or 1800 and 1 mg/kg of vitamin K 8 hr later and the rats were sacrificed 240 min after vitamin K administration. The activities of the vitamin K reductase and vitamin K epoxide reductase were measured indirectly by determining the content of vitamin K1 and vitamin K epoxide reductase in the plasma and liver. The data obtained in control rats indicated that vitamin K and vitamin K 2,3 epoxide concentrations in plasma and liver were higher (P less than 0.05) at 1800 than at 0600. Warfarin had a greater (P less than 0.05) inhibitory effect on the vitamin K and vitamin K-epoxide reductases at 0600 compared to 1800; plasma levels of S- and R-warfarin did not vary with time of administration. The findings suggest that the activity of both reductases under control conditions, and the warfarin-induced inhibition of these enzymes varied depending on the time of drug administration.     

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.     

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.     

Mechanism of coumarin action: sensitivity of vitamin K metabolizing enzymes of normal and warfarin-resistant rat liver

The in vitro effects of two coumarin anticoagulants, warfarin and difenacoum, on rat liver microsomal vitamin K dependent carboxylase, vitamin K epoxidase, vitamin K epoxide reductase, and cytosolic vitamin K reductase (DT-diaphorase) from the livers of normal and a warfarin-resistant strain of rats have been determined. Millimolar concentrations of both coumarins are required to inhibit the carboxylase and epoxidase activities in both strains of rats. Sensitivity of DT-diaphorase to coumarin inhibition differs when a soluble or liposomal-associated substrate is used, but the diaphorases isolated from both strains of rats have comparable sensitivity. The anticoagulant difenacoum is an effective rodenticide in the warfarin-resistant strain of rats, and the only enzyme studied from warfarin-resistant rat liver that demonstrated a significant differential inhibition by the two coumarins used was the vitamin K epoxide reductase. This enzyme also showed the greatest sensitivity to coumarin inhibition among the enzymes studied. These results support the hypothesis that the physiologically important site of action of coumarin anticoagulants is the vitamin K epoxide reductase.