Vitamin K-dependent carboxylase: evidence for a hydroperoxide intermediate in the reaction.

Abstract

Vitamin K is an essential cofactor for a microsomal carboxylase that converts glutamyl residues in endogenous precursor proteins to y-carboxyglutamyl residues in completed proteins. The same microsomal preparations convert vitamin K to its 2,3-epoxide, and it has been suggested that these two reactions (carboxylation and epoxidation) are coupled. Glutathione peroxidase, which reduces hydrogen peroxide and organic hydroperoxides, inhibits both of these reactions in a preparation of microsomes solubilized by Triton X-100. Catalase has no effect. In the absence of vitamin K, and in the presence of NADPH, tert-butyl hydroperoxide acts as a weak vitamin K analog. At lower concentrations, tert-butyl hydroperoxide is an apparent competitive inhibitor of vitamin K for both the carboxylase and epoxidase reactions. These data are consistent with the hypothesis that both of these vitamin K-requiring reactions involve a common oxygenated intermediate, and that a hydroperoxide of the vitamin is the species involved. Vitamin K functions in the postribosomal modification of liver microsomal precursor proteins to form biologically active prothrombin, and the other vitamin K-dependent plasma clotting factors: VII, IX, and X (1, 2). The vitamin K-dependent modification of these precursors involves the carboxylation of specific glutamyl residues to form y-carboxyglutamyl residues in the completed proteins, and in vitro microsomal systems that carry out this vitamin K-dependent carboxylation have been developed (3)(4)(5)(6)(7). These systems, which have now been solubilized (8-13), have utilized the endogenous microsomal precursor protein(s) as a substrate for the carboxylase; more recently, synthetic peptides that are analogous to Glu-containing regions of the prothrombin precursor have been used as substrates (14,15). This carboxylation reaction requires the reduced form of vitamin K, 02, and CO2 but appears to have no ATP, coenzyme A, or biotin requirement. The same microsomal preparations that catalyze the carboxylation also convert vitamin K to its 2,3-epoxide (16-18). It has been suggested (16) that the epoxidation reaction may be coupled to the carboxylation reaction, and the evidence that relates to this hypothesis has recently been reviewed (19). One possible mode of coupling of these two reactions would be through a common intermediate. This report presents evidence that is consistent with the hypothesis that a hydroperoxide of the vitamin is involved in both of these reactions, and suggests possible molecular roles for the vitamin in this unique carboxylase.MATERIALS AND METHODS Incubation Conditions. Microsomal pellets were prepared from 7-to 10-day vitamin K-deficient rat livers as previously described (14). The microsome pellets were solubilized in SIK (0.25 M sucrose/0.025 M imidazole/0.5 M KC1, pH 7.2) buffer containing 1.5% (vol/vol) Triton X-100 and centrifuged at 105,000 X g for 60 min to remove insoluble material. Incubations consisted of 0.4 ml of this supernatant and 0.1 ml of SIK buffer containing NAD...

Warfarin poisoning and vitamin K antagonism in rat and human liver. Design of a system in vitro that mimics the situation in vivo

“…This simple kinetic pattern was also observed by Larson & Suttie (1978) when chemically reduced vitamin K1 was used as cofactor for the carboxylase. It is difficult to envisage a model for this complex enzyme system which can explain the simple enzyme kinetics observed.…”

Poisoning by Anticoagulant Rodenticides in Humans and Animals: Causes and Consequences

“…These factors, called vitamin K-dependent cloting factors, have to go through a post-translational gamma-carboxylation of their glutamate residues into gamma-carboxyglutamic acid to be able to chelate calcium and have their physiological activity [15,16]. This reaction is done by gamma-glutamyl carboxylase (GGCX), which is another membrane enzyme of endoplasmic reticulum, and needs the oxidation of vitamin K hydroquinone to vitamin K epoxide to provide the required reducing power [17,18]. Then VKORC1 recycles vitamin K epoxides to vitamin K hydroquinones (Figure 2) [19].…”

Evaluation of a Modified Enzyme Linked Immunosorbent Assay for Serum PIVKA-II Measurement

“…PIVKA-II는 프로트롬빈의 비정상적인 합성 과정에 서 생성되는 물질로, 정상적으로는 간에서 프로트롬빈 전구물질 의 아미노산 말단(amino terminal)에 10개의 glutamic acid (Glu)가 비타민 K 의존 carboxylase에 의해 γ-carboxyglutamic acid (Gla)로 변환되는 과정을 통해 생성되나 [10] 비타민 K 결핍이 있는 경우에 는 10개의 Glu의 전부 또는 일부가 Gla로 변환되지 못하고 이상프 로트롬빈이 혈액으로 분비될 수 있는데 이것을 PIVKA-II 라고 한 다 [11,12]. 특히 간문맥을 침범하여 진행된 경우 대부분의 환자에 서 PIVKA-II가 증가됨이 보고되어 [13,14] 빠르게 진행하거나 나쁜 예후를 나타내는 표지자로도 유용함이 제시되었다 [15].…”