Proc. Natl. Acad. Sci. USA Vol. 75, No. 11, pp. 5413-5416, November 1978 Biochemistry Vitamin K-dependent carboxylase: Evidence for a hydroperoxide intermediate in the reaction ('y-carboxyglutamic acid/prothrombin/vitamin K epoxide) A. E. LARSON AND J. W. SUTTIE* Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706 Communicated by Henry Lardy, August 28,1978

ABSTRACT Vitamin K is an essential cofactor for a mi- 105,000 X g for 60 min to remove insoluble material. Incuba- crosomal carboxylase that converts glutamyl residues in en- tions consisted of 0.4 ml of this supernatant and 0.1 ml of SIK dogenous precursor proteins to y-carboxyglutamyl residues in buffer containing NADPH, peptide substrate, and other com- completed proteins. The same microsomal preparations convert vitamin K to its 2,3-epoxide, and it has been suggested that these pounds as described in the figure legends. Tert-butyl hydro- two reactions (carboxylation and epoxidation) are coupled. peroxide (t-butyl-OOH) was added directly to the incubation Glutathione peroxidase, which reduces hydrogen peroxide and mixtures. Vitamin K was reduced with sodium dithionite (4), organic hydroperoxides, inhibits both of these reactions in a and vitamin K hydroquinone was added to the incubations in preparation of microsomes solubilized by Triton X-100. Catalase ethanol. Radioactive CO2 was added as 20 ,uCi of NaH'4CO3 has no effect. In the absence of vitamin K, and in the presence per ml (59.3 mCi/mmol, (1 Ci = 3.7 X 1010 of NADPH, tert-butyl hydroperoxide acts as a weak vitamin K Amersham/Searle) analog. At lower concentrations, tert-butyl hydroperoxide is an becquerels). Incubations were carried out at 27°C for 30 min apparent competitive inhibitor of vitamin K for both the car- with shaking in 13 X 100 mm tubes sealed with Parafilm. An- boxylase and epoxidase reactions. These data are consistent with aerobic incubations were flushed with N2 for 30 min before the the hypothesis that both of these vitamin K-requiring reactions 14CO2 and vitamin were added. Glucose oxidase (50 units/ml), involve a common oxygenated intermediate, and that a hydro- glucose (7 mM), and catalase (50 units/ml) were also present peroxide of the vitamin is the species involved. in anaerobic incubations to ensure oxygen depletion (units are Vitamin K functions in the postribosomal modification of liver as defined by the supplier). microsomal precursor proteins to form biologically active Conversion of vitamin K to its 2,3-epoxide (epoxidation) was prothrombin, and the other vitamin K-dependent plasma measured by incubation with [3H]vitamin K, extracting the clotting factors: VII, IX, and X (1, 2). The vitamin K-dependent incubation mixtures with 2 vol of 3:2 (vol/vol) isopropanol/ modification of these precursors involves the carboxylation of hexane, and analyzing the extracts by thin-layer chromatog- specific glutamyl residues to form y-carboxyglutamyl residues raphy as previously described (18). in the completed proteins, and in vitro microsomal systems that Measurement of Radioactivity. Incorporation of "4CO2 into carry out this vitamin K-dependent carboxylation have been protein was measured by using repetitive trichloracetic acid developed (3-7). These systems, which have now been solubi- precipitations and washes as previously described (8), and lized (8-13), have utilized the endogenous microsomal pre- carboxylation of Phe-Leu-Glu-Glu-Leu was determined (14) cursor protein(s) as a substrate for the carboxylase; more re- by measuring fixed CO2 in the trichloroacetic acid-soluble cently, synthetic peptides that are analogous to Glu-containing material.t One milliliter of 10% trichloracetic acid was added regions of the prothrombin precursor have been used as sub- to a 0.2-ml aliquot of the incubation mixture. This was imme- strates (14, 15). This carboxylation reaction requires the reduced diately mixed with a vortex stirrer and allowed to sit at least 15 form of vitamin K, 02, and CO2 but appears to have no ATP, min on ice. The precipitate was centrifuged, and excess 14CO2 coenzyme A, or biotin requirement. The same microsomal was removed from the supernatant by gassing with CO2 for 5 preparations that catalyze the carboxylation also convert vita- min. A 0.2-ml aliquot of the degassed supernatant was added min K to its 2,3-epoxide (16-18). It has been suggested (16) that to 4.3 ml of Aquasol and shaken, and radioactivity was deter- the epoxidation reaction may be coupled to the carboxylation mined in a liquid scintillation spectrometer with external reaction, and the evidence that relates to this hypothesis has standardization. Counting efficiency was 70%. recently been reviewed (19). One possible mode of coupling Enzymes and Chemicals. Glutathione peroxidase was pu- of these two reactions would be through a common interme- rified from rat liver according to the method of Nakamura et diate. This report presents evidence that is consistent with the al. (20) to a specific activity of 73.3 units/mg (one unit = 1 ,tmol hypothesis that a hydroperoxide of the vitamin is involved in of H202 reduced per min at 25°C). A single band was obtained both of these reactions, and suggests possible molecular roles upon electrophoresis in nondenaturing 7% and 9% polyacryl- for the vitamin in this unique carboxylase. amide gels. Glutathione reductase, catalase (C-100, twice re- MATERIALS AND METHODS Abbreviations: t-butyl-OOH, tert-butyl hydroperoxide; GSH-Px, glutathione peroxidase. Incubation Conditions. Microsomal pellets were prepared * To whom reprint requests should be addressed. from 7- to 10-day vitamin K-deficient rat livers as previously t Peptide carboxylase activity refers to the amount of vitamin K- described (14). The microsome pellets were solubilized in SIK dependent nonvolatile radioactivity present in a trichloroacetic acid (0.25 M sucrose/0.025 M imidazole/0.5 M KC1, pH 7.2) buffer supernatant after incubation in the presence of a peptide substrate and H14CO3-. Protein carboxylase activity refers to the vitamin containing 1.5% (vol/vol) Triton X-100 and centrifuged at K-dependent trichloroacetic acid-precipitable radioactivity in the same system. The endogenous protein substrates carboxylated include The publication costs of this article were defrayed in part by page the prothrombin precursors (3, 8) and presumably precursors of other charge payment. This article must therefore be hereby marked "ad- vitamin K-dependent proteins. It has been previously shown (8, 14) vertisement" in accordance with 18 U. S. C. §1734 solely to indicate that the radioactivity fixed in both systems is present as -y-carboxy- this fact. glutamic acid. 5413 Downloaded by guest on September 27, 2021 5414 Biochemistry: Larson and Suttie Proc. Natl. Acad. Sci. USA 75 (1978)

crystallized), glucose oxidase, glutathione, and vitamin K1 were Table 1. t-Butyl-OOH stimulation of peptide and from Sigma. Aquasol and Econofluor were from New England protein carboxylation Nuclear (Boston, MA); NCS was from Amersham/Searle. Triton Peptide Protein X-100 was from Research Products International (Elk Grove, Additions to % % IL) and was scintillation grade. All other chemicals were reagent incubation grade. [3H]Vitamin K1 was synthesized (21) from tetrasodium dpm/ml change dpm/ml change 2-methyl[5,6,7,8-3H]naphtha-1,4-quinol diphosphate (80 Ci/ None 1660 + 201 1058 ± 121 mmol, Amersham/Searle) to a specific activity of 5.3 Ci/mmol. t-Butyl-OOH Peptide substrate, Phe-Leu-Glu-Glu-Leu, was obtained from (5 Al) 2472 164 +48.9 1694 i114 +60.0 Vega-Fox Biochemicals (Tucson, AZ). t-Butyl-OOH (10lg) 2611 +57.2 1753 +65.6 RESULTS t-Butyl-OOH Effect of Glutathione Peroxidase and Catalase. A logical (5 Al) + in the formation of the 2,3-epoxide of vitamin K GSH-Px intermediate (3 units/ml) 1632 -1.6 1112 +4.8 would be the 2- or 3-hydroperoxide of the vitamin. Glutathione peroxidase (GSH-Px), which will reduce organic hydroperox- All incubations contained 1 mM NADPH, reduced vitamin K at ides to the corresponding alcohols (22), was therefore tested as 50 gg/ml, and 0.5 mM peptide. The GSH-Px incubation had gluta- an inhibitor of carboxylation and epoxidation in a Triton-sol- thione and additions as described in Fig. 1. Values are averages of five ubilized microsomal system. The data in Fig. 1 illustrate that incubations ± SD or average of duplicate incubations. both protein and peptide carboxylation and vitamin K epoxide formation were completely inhibited by the addition of GSH-Px a 20% inhibition of both reactions at an H202 concentration of to the incubation at 3 units/ml. In addition, partial inhibition 10mM. of the carboxylation and epoxidation reactions demonstrated t-Butyl-OOH as a Vitamin K Analog. The possible in- a similar dependence on increasing GSH-Px concentration. This volvement of the hydroperoxide of vitamin K as an important of GSH-Px caused the intermediate suggested that stable organic hydroperoxides, such inhibition suggested that the addition re- reduction of a vitamin K hydroperoxide intermediate essential as t-butyl-OOH, might be able to drive the carboxylation for both epoxide formation and carboxylation. The typical action. In the absence of vitamin K, t-butyl-OOH was found to stimulate CO2 fixation into both peptide substrate and en- flavoprotein oxidation of a reduced quinone would be expected was abolished by to yield H202. GSH-Px will also utilize H202 as a substrate; dogenous protein (Table 1). This stimulation therefore, to determine if the effect of GSH-Px could have been the addition of GSH-Px to the incubation media. This stimu- of H202, the effect of catalase addition lation of carboxylation seen under these conditions was low, through the destruction about 60% above background, but was reproducibly seen and to the incubation was also investigated. As seen in Fig. 1, cata- was statistically significant. To determine if the fixed CO2 was lase no on or formation. had effect either carboxylation epoxide into acid, the peptide product a for- incorporated y-carboxyglutamic Further evidence that hydronaphthoquinone-mediated in acid supernatant was partially purified not in reaction was obtained by the trichloroacetic mation of H202 is involved the by Sephadex G-25 and Bio-Gel P-2 chromatography as previ- assessing the effect of added H202 on the vitamin K-dependent ously described (14). Malonic acid derivatives are unstable reactions. The addition of a low concentration of H202 (10 nM) under acidic conditions, and 'y-carboxyglutamic will decar- did not stimulate either reaction, but inhibited both carboxy- boxylate under conditions normally used for peptide hydrolysis lation and epoxidation by about 10%. Increasing the concen- (1, 2). It was found that 56% of the radioactivity in the crude tration of H202 added to the incubation medium resulted in peptide preparations was lost upon acid hydrolysis. The re- maining radioactivity cochromatographed with glutamic acid A BA on silicic acid thin-layer plates (14). After alkaline hydrolysis

100 * * 100 I (14), the radioactivity cochromatographed with authentic -y- carboxyglutamic acid in the same system. t80 800 The majority of the fixed radioactivity in the nonstimulated 260 incubations is CO2 nonspecifically incorporated into com- 'O 60 60- pounds other than y-carboxyglutamic acid. It is possible to isolate the carboxylated peptide product in a pure form by 0C 40 40 chromatography on DEAE-Sephadex (J. Finnan and J. W. Suttie, unpublished data). When this is done, the apparent stimulation of the carboxylase by t-butyl-OOH increases drastically (Table 2). From the perchloric acid-soluble fraction of an incubation that had an 8000 dpm/ml activity in a blank [Enzyme], units/ml and 13,300 dpm/ml in an incubation with t-butyl-OOH, it was possible to isolate products that had 450 and 4410 dpm/ml in FIG. 1. Effect of GSH-Px and catalase on vitamin K-dependent the corresponding fractions. The true stimulation of incorpo- carboxylation and epoxidation. Incubation mixtures with GSH-Px ration of CO2 into y-carboxyglutamic acid was, therefore, contained purified enzyme, 2 mM glutathione, glutathione reductase nearly 10-fold rather than the 60% seen in the crude prepara- at 2 units/ml, 1 mM EDTA, NADPH at 1 mg/ml, and 0.5 mM peptide. Vitamin was tritiated vitamin K hydroquinone at 10 ,ug/ml, and car- tion. This is, however, still much lower than the stimulation that boxylation and epoxidation were determined on aliquots of the same can be seen with the vitamin. Cumene hydroperoxide had about incubation. Catalase incubations had no GSH-Px, glutathione, EDTA, 70% the activity of t-butyl-OOH in this system. or glutathione reductase. Vitamin K hydroquinone was at 20,g/ml The data in Table 3 provide additional evidence that t- for carboxylation and 4 ug/ml for epoxidation assays. (A) Vitamin butyl-OOH has a specific role in this reaction rather than acting K-dependent carboxylase activity. *, Protein, and 0, peptide car- as a nonspecific oxidant that causes Y-carboxyglutamic acid boxylase activity in the presence of GSH-Px. A, Peptide carboxylase activity in the presence of catalase. (B) Vitamin K-epoxidase activity. formation in a mechanism not related to the vitamin K-stim- 0, In the presence of GSH-Px; *, in the presence of catalase. ulated activity. The vitamin K-dependent carboxylase requires Downloaded by guest on September 27, 2021 Biochemistry: Larson and Suttie Proc. Natl. Acad. Sci. USA 75 (1978) 5415 Table 2. Isolation of carboxylated peptide Stage of dpm/ml purification -t-Butyl-OOH +t-Butyl-OOH Perchlorate supernatant 8000 13,300 Sephadex G-25 6273 10,900 E DEAE-Sephadex A-25 450 4,410 Two milliliters of incubated solubilized microsomes were precipi- tated with 0.17 ml of 60% perchloric acid. After neutralization with K2CO3 and centrifugation, the material was applied to a 1 X 50 cm Sephadex G-25 column. This pool was lyophilized and applied to a 2 1 X 20 cm DEAE-Sephadex A-25 column in 0.01 M ammonium ace- tate and eluted with a 0.01-0.25 M ammonium acetate gradient. Seventy percent of the radioactivity in the final preparation was re- covered with a y-carboxyglutamic acid standard on thin-layer chro- 0 0.02 0.04 0.06 0.08 0.10 0.4 0.8 1.2 1.6 2.0 matography after basic hydrolysis. 1/[S], (jig/ml KH2)-' FIG. 2. Double reciprocal plots of vitamin K-dependent car- if is utilized to form a the boxylase (A) and vitamin K-epoxidase (B) activities in the presence 02 (8); oxygen being hydroperoxide, (U) and absence (0) of t-butyl-OOH. Incubations were started with t-butyl-OOH-driven carboxylation should not require 02. The vitamin K hydroquinQne (KH2) and ran for 20 min, at which time both data in Table 3 indicate that the vitamin K-dependent car- reactions were still linear with respect to time. The concentration of boxylase was inhibited 93% by anaerobic conditions, but the t-butyl-OOH was 10 mM and of peptide substrate, 0.5 mM. KO, vi- t-butyl-OOH-stimulated incorporation was actually enhanced. tamin K 2,3-epoxide. Apparent Ki values were 10 mM for epoxidation A second characteristic of this system that supports the signif- and 2.5 mM for carboxylation. icance of the hydroperoxide-stimulated carboxylation is the specific requirement for NADPH. In the absence of NADPH, t-butyl-OOH did not stimulate the formation of -y-carboxy- butyl-OOH would be consistent with the general hydrophobic glutamic acid. The requirement for NADPH could not be character of the proposed hydroperoxide moiety being the only satisfied by equimolar concentrations of NADH or di- feature common to vitamin K and t-butyl-OOH. The com- thiothreitol. Ultraviolet irradiation sufficient to destroy the petitive inhibition of t-butyl-OOH on the vitamin K-driven amount of vitamin normally added to the microsomes had no carboxylation and vitamin K-epoxide formation suggests that it is acting at the same site as some intermediate of the vitamin. effect on the t-butyl-OOH-dependent carboxylation, indicating The that t-butyl-OOH was not interacting with residual vitamin in NADPH requirement enforces the belief that the CO2 7 fixation seen is not artifactual. The formation of vitamin K- the to 10-day deficient liver microsomes. epoxide from vitamin K hydroquinone and 02 is an internal Although, in the absence of the vitamin, t-butyl-OOH at monooxygenase reaction; and, if this formation is related to the concentrations of from 0.05 to 0.1 M appeared to act as a weak carboxylation reaction, it is likely that reducing equivalents analog of the vitamin, these and lower concentrations of hy- from other sources would be needed to enable t-butyl-OOH droperoxide were found to inhibit both the vitamin K-epoxidase to drive the carboxylation. One hypothesis for epoxide forma- activity and the vitamin K-dependent carboxylase activity. tion (24) has been that H202 generated from the oxidation of When this response was studied in more detail, it was seen (Fig. the vitamin hydroquinone would be used to form the epoxide. 2) that t-butyl-OOH was acting as an apparent competitive The lack of inhibition by catalase in this system would tend to inhibitor of both of these vitamin K-dependent activities. argue against this possibility unless it is assumed that an ex- tremely tight and specifically enzyme-bound H202 is gener- DISCUSSION ated. These data do not suggest any unequivocal molecular role The ability of the enzyme glutathione peroxidase, which has for vitamin K during the carboxylation. Epoxide formation previously been shown to destroy hydroperoxide intermediates from a vitamin K hydroperoxide could generate a basic oxygen in enzyme reactions (23), to inhibit both the vitamin K-de- species that would then act to abstract the y proton of the glu- pendent carboxylase and the vitamin K-epoxidase activity of tamyl residues, leaving a carbanion for CO2 attack. Such a re- rat liver microsomes strongly suggests a hydroperoxide inter- action would undoubtedly require neutralization of the nega- mediate in these reactions. The activity of the organic hydro- tively charged carboxylate ion by the enzyme and would not peroxide t-butyl-OOH (which has little structural similarity explain the NADPH requirement. It is also conceivable that the to vitamin K) in the carboxylation reaction supports this hy- hydroperoxide would be used to form a peracid ester of the pothesis. The requirement for a high concentration of t- glutamyl residue to be carboxylated, or that a vitamin hydro- peroxide is acting in some manner to activate the CO2. It is Table 3. Effect of gas phase on carboxylation possible that the active species could be a hydroperoxide radical and that hydrogen removal via a radical mechanism may be dpm/ml involved in the mechanism. Heme centers, such as the cyto- Additions to 02 N2 chrome P-450 system, can act as peroxidases (25), and t- incubation atmosphere atmosphere butyl-OOH may be acting to oxygenate a heme center that can None 2,115 2265 then act to oxidatively remove the glutamyl -y proton. In any t-Butyl-OOH 3,105 5040 event, the formation of an epoxide from a hydroperoxide is Vitamin K hydroquinone chemically favorable, and it is reasonable to consider using this (50,gg/ml) 12,862 energy to at least partially drive the carboxylation reaction. 3060 Although the observations reported here do not assign a par- Anaerobic incubations were carried out in sealed vials that had been ticular role to the vitamin in the unique carboxylation reaction, gassed with N2 for 30 min before incubation. Glucose oxidase at 50 they do suggest possible roles of the vitamin in driving the re- units/ml, 7 mM glucose, and catalase at 50 units/ml were also present action and must be considered in any hypothesis of vitamin K to remove residual 02- Values are averages ofduplicate incubations. action. Downloaded by guest on September 27, 2021 5416 Biochemistry: Larson and Suttie Froc. Natt. Acad. Sci. USA 75 (1978)

This research was supported by the College of Agricultural and Life 13. Vermeer, C., Soute, B. A. M. & Hemker, H. C. (1978) Biochim. Sciences, University of Wisconsin-Madison, and in part by U.S. Public Biophys. Acta 523, 494-505. Health Service National Institutes of Health Grant AM-14881 and 14. Suttie, J. W., Hageman, J. M., Lehrman, S. R. & Rich, D. H. National Institutes of Health predoctoral training Grant GM-07215. (1976) J. Biol. Chem. 251, 5827-5830. 15. Houser, R. M., Carey, D. J., Dus, K. M., Marshall, G. R. & Olson, 1. Suttie, J. W. & Jackson, C. M. (1977) Physiol. Rev. 57, 1-70. R. E. (1977) FEBS Lett. 75,226-230. 2. Stenflo, J. & Suttie, J. W. (1977) Annu. Rev. Biochem. 46, 16. Willingham, A. K. & Matschiner, J. T. (1974) Biochem. J. 140, 157-172. 435-441. 3. Esmon, C. T., Sadowski, J. A. & Suttie, J. W. (1975) J. Biol. Chem. 17. Bell, R. G. & Stark, P. (1976) Biochem. Biophys. Res. Commun. 250,4744-4748. 72, 619-625. 4. Sadowski, J. A., Esmon, C. T. & Suttie, J. W. (1976) J. Biol. Chem. 18. Sadowski, J. A., Schnoes, H. K. & Suttie, J. W. (1977) Biochemistry 16,3856-3863. 251,2770-2775. 19. Suttie, J. W., Larson, A. E., Canfield, L. M. & Carlisle, T. L. (1978) 5. Girardot, J.-M., Mack, D. O., Floyd, R. A. & Johnson, B. C. (1976) Fed. Proc. Fed. Am. Soc. Exp. Biol. 37, in press. Biochem. Biophys. Res. Commun. 70,655-662. 20. Nakamura, W., Hosoda, S. & Hayashi, K. (1974) Biochim. Bio- 6. Friedman, P. A. & Shia, M. (1976) Biochem. Biophys. Res. phys. Acta 358,251-261. Commun. 70, 647-654. 21. Matschiner, J. T. (1970) in The Fat-Soluble Vitamins, eds. De- 7. Helgeland, L. (1977) Biochim. Biophys. Acta 499, 181-193. Luca, H. F. & Suttie, J. W. (Univ. Wisconsin Press, Madison, WI), 8. Esmon, C.. T. & Suttie, J. W. (1976) J. Biol. Chem. 251, 6238- p. 377. 6243. 22. Burk, R. F., Katsuyuki, N., Lawrence, R. A. & Chance, B. (1978) 9. Mack, D. O., Suen, E. T., Girardot, J.-M., Miller, J. A., Delaney, J. Biol. Chem. 253,43-46. R. & Johsnon, B. C. (1976) J. Biol. Chem. 251, 3269-3276. 23. Smith, W. L. & Lands, W. E. M. (1972) Biochemistry 11, 10. Jones, J. P., Gardner, E. J., Cooper, T. G. & Olson, R. E. (1977) 3276-3285. J. Biol. Chem. 252,7738-7742. 24. Olson, R. E. & Suttie, J. W. (1978) Vitam. Horm. (NY) 35,59- 11. Friedman, P. A. & Shia, M. A. (1977) Biochem. J. 163,39-43. 108. 12. Wallin, R., Gebhardt, 0. & Prydz, H. (1978) Biochem. J. 169, 25. Hrycay, E. G., Jonen, H. G., Lu, A. Y. H. & Levin, W. (1975) 95-101. Arch. Biochem. Biophys. 166, 145-151. Downloaded by guest on September 27, 2021