The Vitamin KDependent Carboxylase

The vitamin K-dependent carboxylase is an integral membrane protein. Most of the proteins that are carboxylated are extracellular proteins, and the major activity of the carboxylase is at the luminal face of the rough endoplasmic reticulum. However, there is also significant carboxylase activity in mitochondria.

As shown in Figure 5.2, the initial reaction is oxidation of vitamin K hydro-quinone to the epoxide, linked to y -deprotonation of the glutamate residue to yield a carbanion, catalyzed by vitamin K epoxidase.

Menaquinones 2 to 7 have essentially the same activity as phylloquinone when the reduced hydroquinones are incubated with either isolated preparations of epoxidase or intact microsomes. If intact microsomes are incubated with the quinones, menaquinone-3 has higher activity than phylloquinone, which in turn has higher activity than menaquinone-4 or higher homologs, suggesting that quinone reductase (see below) has greater specificity for the length of the side chain than does the epoxidase. The glutamate

vitamin K quinone

Figure 5.2. Reaction of the vitamin K-dependent carboxylase (vitamin K epoxidase) and recycling of vitamin K epoxide to the hydroquinone. Vitamin K epoxidase, EC; warfarin-sensitive epoxide/quinone reductase, EC; and warfarin-insensitive quinone reductase, EC

vitamin K quinone

Figure 5.2. Reaction of the vitamin K-dependent carboxylase (vitamin K epoxidase) and recycling of vitamin K epoxide to the hydroquinone. Vitamin K epoxidase, EC; warfarin-sensitive epoxide/quinone reductase, EC; and warfarin-insensitive quinone reductase, EC

carbanion formed by the epoxidase reaction reacts with carbon dioxide to form Y-carboxyglutamate. At saturating concentrations of carbon dioxide, there is equimolar formation of y-carboxyglutamate and vitamin K epoxide.

The double bond at C-3 of the side chain is essential to activity and has frans-configuration; ds-phylloquinone has no biological activity. This double bond seems to be involved in the deprotonation of the glutamate substrate.

Target proteins for carboxylation have the recognition sequence X-Phe-X-aa-aa-aa-aa-Ala, where X is an aliphatic hydrophobic amino acid, and 10 to 12 glutamate residues in the first 40 amino acids of the amino terminal region. The peptide substrate binds to the amino terminal region of the carboxylase, whereas the carboxyl terminal region has the epoxidase activity. The epoxidase activity is regulated by the binding of the peptide substrate and in the absence of the peptide, epoxidation does not occur (Furie and Furie, 1997; Sugiura et al., 1997). The enzyme catalyzes multiple carboxylation of glutamate groups, each associated with epoxidation of vitamin K hydroquinone, during a single peptide binding event; no partially carboxylated peptide is released from the enzyme to be rebound and undergo further carboxylation, although in vitamin K deficiency partially carboxylated peptides are released (Morris et al., 1995).

Vitamin K epoxide is reduced to the quinone in a reaction involving oxidation of a dithiol to the disulfide, catalyzed by epoxide reductase. This enzyme has no activity toward menadione epoxide or the epoxides of a variety of xeno-biotics, but is specific for alkylated vitamin K epoxides. Like other dithiol-linked flavoprotein reductases, this enzyme initially undergoes an internal dithiol-disulfide reaction, followed by reaction with the dithiol substrate. The initial step in the reaction is an attack on the epoxide ring by an enzyme-bound sulfhydryl group, with intermediate formation of a thioether adduct. Thiol reagents, such as iodoacetamide and N-ethylmaleimide, inhibit the enzyme irreversibly. The addition of vitamin K epoxide protects the enzyme against this inactivation. The physiological dithiol substrate has not been uniquevocally identified, but is assumed to be thioredoxin.

Vitamin K quinone is reduced to the active hydroquinone substrate for the epoxidase reaction by either a dithiol-linked reductase that is almost certainly the same enzyme as the epoxide reductase or NADPH-dependent quinone reductase. Like the epoxide reductase, the dithiol-linked reductase is inhibited by warfarin. In warfarin-resistant rats, there is a warfarin-insensitive epoxide reductase, which also has quinone reductase activity (Hildebrandt et al., 1984; Gardill and Suttie, 1990).

The NADPH-dependent reduction of vitamin K quinone to the hydro-quinone is not inhibited by warfarin. In the presence of adequate amounts of vitamin K, the carboxylation of glutamate residues can proceed normally, despite the presence of warfarin, with the stoichiometric formation of vitamin K epoxide that cannot be reutilized. Small amounts of vitamin K epoxide, and hydroxides formed by its reduction by other enzymes, are normally found in plasma. In warfarin-treated animals and patients, there is a significant increase in the plasma concentration of both. There is also an increase in the urinary excretion of the products of side-chain oxidation of the epoxide and hydroxides.

Prothrombin normally contains 10 y-carboxyglutamate residues in the amino terminal region. In the presence of high concentrations of warfarin, a completely uncarboxylated precursor, preprothrombin, is released into the circulation. Before the nature of this precursor protein was known, it was called protein induced by vitamin K absence or antagonism (PIVKA), a term that is sometimes still used.

At lower doses of anticoagulant, a variety of partially carboxylated pre-prothrombins are formed. Sequencing of these proteins shows that the carboxylation is not random, but proceeds in an orderly fashion from the amino terminal of the substrate. In preprothrombin containing 80% of the normal amount of y -carboxyglutamate, it is the last two glutamate residues before the carboxy terminal that are not carboxylated; in 60% undercarboxylated pre-prothrombin, it is the last four glutamate residues that are not carboxylated (Liska and Suttie, 1988).

In some patients with combined deficiency of vitamin K-dependent coagulation factors, the deficiency can be partially corrected by high doses of vitamin K, suggesting that the defect is in the affinity of the carboxylase for its coenzyme (Mutucumarana et al., 2000).

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