Vitamin K-Dependent Carboxylase

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 (3-7). 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 7to 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 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. ?1734 solely to indicate this fact. 54 Evidence for a hydroperoxide y of Wisconsin, Madison, Wisconsin 53706 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 NADPH, peptide substrate, and other compounds as described in the figure legends. Tert-butyl hydroperoxide (t-butyl-OOH) was added directly to the incubation mixtures. Vitamin K was reduced with sodium dithionite (4), and vitamin K hydroquinone was added to the incubations in ethanol. Radioactive CO2 was added as 20 jCi of NaH14CO3 per ml (59.3 mCi/mmol, Amersham/Searle) (1 Ci = 3.7 X 1010 becquerels). Incubations were carried out at 27?C for 30 min with shaking in 13 X 100 mm tubes sealed with Parafilm. Anaerobic incubations were flushed with N2 for 30 min before the "4CO2 and vitamin were added. Glucose oxidase (50 units/ml), glucose (7 mM), and catalase (50 units/ml) were also present in anaerobic incubations to ensure oxygen depletion (units are as defined by the supplier). Conversion of vitamin K to its 2,3-epoxide (epoxidation) was measured by incubation with [3H]vitamin K, extracting the incubation mixtures with 2 vol of 3:2 (vol/vol) isopropanol/ hexane, and analyzing the extracts by thin-layer chromatography as previously described (18). Measurement of Radioactivity. Incorporation of 14CO2 into protein was measured by using repetitive trichloracetic acid precipitations and washes as previously described (8), and carboxylation of Phe-Leu-Glu-Glu-Leu was determined (14) by measuring fixed CO2 in the trichloroacetic acid-soluble material.t One milliliter of 10% trichloracetic acid was added to a 0.2-ml aliquot of the incubation mixture. This was immediately mixed with a vortex stirrer and allowed to sit at least 15 min on ice. The precipitate was centrifuged, and excess 14C02 was removed from the supernatant by gassing with CO02 for 5 min. A 0.2-ml aliquot of the degassed supernatant was added to 4.3 ml of Aquasol and shaken, and radioactivity was determined in a liquid scintillation spectrometer with external standardization. Counting efficiency was 70%. Enzymes and Chemicals. Glutathione peroxidase was purified from rat liver according to the method of Nakamura et al. (20) to a specific activity of 73.3 units/mg (one unit = 1 jmol of H202 reduced per min at 25?C). A single band was obtained upon electrophoresis in nondenaturing 7% and 9% polyacrylamide gels. Glutathione reductase, catalase (C-100, twice reAbbreviations: t-butyl-OOH, tert-butyl hydroperoxide; GSH-Px, glutathione peroxidase. * To whom r print requests should be addressed. t Peptide carboxylase activity refers to the amount of vitamin Kdependent nonvolatile radioactivity present in a trichloroacetic acid supernatant after incubation in the presence of a peptide substrate and H"4CO-. Protein carboxylase activity refers to the vitamin K-dependent trichloroacetic acid-precipitable radioactivity in the same system. The endogenous protein substrates carboxylated include the prothrombin precursors (3, 8) and presumably precursors of other vita in K-dependent proteins. It has been previously shown (8, 14) that the rad oact vity fixed in both systems is present as 'y-carboxyglutamic acid.