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.     

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.     

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.     

Apoptosis-inducing activity of vitamin C and vitamin K.

Apoptosis-inducing activity of vitamins C and K and of their analogs are reviewed. Vitamin C shows both reducing and oxidizing activities, depending on the environment in which this vitamin is present. Higher concentrations of vitamin C induce apoptotic cell death in various tumor cell lines including oral squamous cell carcinoma and salivary gland tumor cell lines, possibly via its prooxidant action. The apoptosis-inducing activity of ascorbate is stimulated by Cu2+, lignin and ion chelator, and inhibited by catalase, Fe3+, Co2+ and saliva. On the other hand, at lower concentrations, ascorbic acid displays an antioxidant property, preventing the spontaneous and stress or antitumor agent-induced apoptosis. Sodium 5,6-benzylidene-L-ascorbate, intravenous administration of which induces degeneration of human inoperable tumors and rat hepatocellular carcinoma in vivo, induces apoptotic or non-apoptotic cell death, depending on the types of target cells. On the other hand, elevation of intracellular concentration of ascorbic acid by treatment with ascorbate 2-phosphate or dehydroascorbic acid makes the cells resistant to the oxidative stress-induced apoptosis. Vitamin K2, which has a geranylgeranyl group as a side chain,and vitamin K3 induces apoptosis of various cultured cells including osteoclasts and osteoblasts, by elevating peroxide and superoxide radicals. Synergistic apoptosis-inducing actions have been found between vitamins C and K, and between these vitamins and antiproliferative agents. The possible therapeutic application of these vitamins is discussed.     

Oxygen dependence of vitamin K-dependent carboxylase and vitamin K epoxidase

A study of the oxygen requirements of the rat liver microsomal vitamin K-dependent carboxylase and vitamin K 2,3-epoxidase indicated that both enzymes had a Km for O2 in the range 60-80 microM. This value was not influenced by vitamin concentration, alterations in carboxylase substrate, Mn2+, or dithiothreitol, and is consistent with the hypothesis that both activities are catalyzed by the same enzyme.     

Evidence for vitamin K semiquinone as the functional form of vitamin K in the liver vitamin K-dependent protein carboxylation reaction.

The protein carboxylating system derived from vitamin K-deficient rat liver microsomes functions in detergent solution if vitamin K₁, NADH, dithiothreitol, CO₂ and O₂ are added. The requirements for added NADH, dithiothreitol and O₂ are all eliminated by the use of vitamin K₁ hydroquinone in place of quinone. The use of the hydroquinone gives a more rapid reaction and a higher yield than does the quinone plus reducing system. The reaction proceeding from either the vitamin K₁ quinone or hydroquinone is blocked by the spin-trapping agent, 5,5-dimethyl-l-pyrroline-N-oxide, suggesting that the active form of vitamin K is the semiquinone.     

Responses of renal and hepatic vitamin K dependent gamma-glutamyl carboxylase substrates to warfarin and vitamin K treatments

A single intraperitoneal injection of warfarin (5 mg/kg) in the rat causes maximal accumulation of hepatic vitamin K dependent carboxylase substrate by 8 hr. In the kidney accumulation is slower with maximal amounts of the substrate appearing at about 16 hr. Vitamin K administered intravenously to warfarin-treated rats causes the complete disappearance of the hepatic substrate in 2 hr. In contrast, neither a single nor multiple injections of the vitamin decrease the renal substrate level by more than 30%. However, this decrease can be augmented by inhibition of protein synthesis with cycloheximide.     

Structural and functional insights into human vitamin K epoxide reductase and vitamin K epoxide reductase-like1

Abstract

Human vitamin K epoxide reductase (hVKOR) is a small integral membrane protein involved in recycling vitamin K. hVKOR produces vitamin K hydroquinone, a crucial cofactor for γ-glutamyl carboxylation of vitamin K dependent proteins, which are necessary for blood coagulation. Because of this, hVKOR is the target of a common anticoagulant, warfarin. Spurred by the identification of the hVKOR gene less than a decade ago, there have been a number of new insights related to this protein. Nonetheless, there are a number of key issues that have not been resolved; such as where warfarin binds hVKOR, or if human VKOR shares the topology of the structurally characterized but distantly related prokaryotic VKOR. The pharmacogenetics and single nucleotide polymorphisms of hVKOR used in personalized medicine strategies for warfarin dosing should be carefully considered to inform the debate. The biochemical and cell biological evidence suggests that hVKOR has a distinct fold from its ancestral protein, though the controversy will likely remain until structural studies of hVKOR are accomplished. Resolving these issues should impact development of new anticoagulants. The paralogous human protein, VKOR-like1 (VKORL1) was recently shown to also participate in vitamin K recycling. VKORL1 was also recently characterized and assigned a functional role as a housekeeping protein involved in redox homeostasis and oxidative stress with a potential role in cancer regulation. As the physiological interplay between these two human paralogs emerge, the impacts could be significant in a number of diverse fields from coagulation to cancer.