Evidence for a Hydroperoxide Intermediate in the Reaction ('Y-Carboxyglutamic Acid/Prothrombin/Vitamin K Epoxide) A
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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.