©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mutagenesis of Vitamin K-dependent Carboxylase Demonstrates a Carboxyl Terminus-mediated Interaction with Vitamin K Hydroquinone (*)

(Received for publication, November 17, 1994)

David A. Roth (§) Michelle L. Whirl Leonardo J. Velazquez-Estades Christopher T. Walsh (1) Bruce Furie Barbara C. Furie

From the Center for Hemostasis and Thrombosis Research, Division of Hematology/Oncology, New England Medical Center and the Departments of Medicine and Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The -glutamyl carboxylase and vitamin K epoxidase activities of a series of mutants of bovine vitamin K-dependent carboxylase with progressively larger COOH-terminal deletions have been analyzed. The recombinant wild-type (residues 1-758) and mutant protein carboxylases, Cbx 711, Cbx 676, and Cbx 572, representing residues 1-711, 1-676, and 1-572, respectively, were expressed in baculovirus-infected Sf9 cells. Wild-type carboxylase had a K for the substrate Phe-Leu-Glu-Glu-Leu (FLEEL) of 0.87 mM; the carboxylation of FLEEL was stimulated 2.5-fold by proPT18, the propeptide of prothrombin. Its K for vitamin K hydroquinone was 23 µM and the specific epoxidase activity of the carboxylase was 938 pmol vitamin KO/30 min/pmol of carboxylase. Cbx 711, which was also stimulated by proPT18, had a K for FLEEL, a K for vitamin K hydroquinone, and a specific epoxidase activity that was comparable to the wild-type carboxylase. In contrast Cbx 572 lacked both carboxylase and epoxidase activities. Although Cbx 676 had a normal carboxylase active site in terms of the K for FLEEL and its stimulation by proPT18, the K for vitamin K hydroquinone was 540 µM, and the specific epoxidase activity was 97 pmol KO/30 min/pmol of Cbx 676. The catalytic efficiencies of Cbx 676 for glutamate carboxylation and vitamin K epoxidation were decreased 15- and 400-fold, respectively, from wild-type enzyme reflecting the requirement for formation of an activated vitamin K species for carboxylation to occur. These data indicate that the truncation of COOH-terminal segments of the carboxylase had no effect on FLEEL or propeptide recognition, but in the case of Cbx 676, selectively affected the interaction with vitamin K hydroquinone and the generation of epoxidase activity. These data suggest that a vitamin K epoxidase activity domain may reside near the COOH terminus while the carboxylase active site domain resides toward the NH(2) terminus.


INTRODUCTION

Vitamin K-dependent -glutamyl carboxylase catalyzes the post-translational modification of glutamate to -carboxyglutamate (Gla) (^1)in the vitamin K-dependent proteins which include several blood clotting proteins (prothrombin, factors VII, IX, and X) and coagulation regulatory proteins (protein C and protein S) (Furie and Furie, 1988). The Gla residues located near the amino terminus of the mature blood clotting proteins facilitate the coordination of calcium ions which in turn stabilizes the tertiary structure of the Gla domain, as exemplified by the crystal structure of bovine prothrombin fragment 1:calcium (Soriano-Garcia et al., 1992). This structure is required to facilitate membrane binding properties necessary for the biological activity of these proteins (Furie and Furie, 1988).

The carboxylase is unique in its requirement for vitamin K. The enzyme uses the reduced form of the vitamin to carboxylate the -carbon of glutamate residues in an oxygen-dependent reaction (Suttie, 1985). Products of the reaction are vitamin K 2,3-epoxide (KO), Gla and H(2)O. The mechanism of carboxylase activity is under intense study. A recently proposed basicity enhancement model (Ham and Dowd, 1990) supported by ^18O(2)-labeling experiments (Dowd et al., 1992; Kuliopulos et al., 1992b) proposes that CO(2) fixation is a late-stage reaction-terminating event which follows O(2)-dependent enzyme catalyzed formation of vitamin K 2,3-epoxide from vitamin K hydroquinone. The hypothesis that the epoxidation of vitamin K and carboxylation of glutamate are carried out by the same enzyme, based on studies with crude enzyme (Suttie, 1985), has been confirmed with purified bovine liver carboxylase (Morris et al., 1993). While these reactions are stoichiometrically coupled when the enzyme is assayed under saturating concentrations of bicarbonate or glutamate-containing substrate, it has been demonstrated that when CO(2) concentrations are reduced below saturation or when glutamyl substrate concentrations are low, vitamin K 2,3-epoxide formation and carboxylation are uncoupled; vitamin K 2,3-epoxide formation proceeds without carboxylation (Larson et al., 1981; Wood and Suttie, 1988). While the chemistry underlying the epoxidation of vitamin K hydroquinone is not fully understood, the carboxylase may be mechanistically related to recently described microbial hydroquinone epoxidases (Gould and Shen, 1991). The recent purification of these soluble enzymes reveals low molecular weight (16,000-22,300) subunit-containing proteins which have hydroquinone epoxidase activity (Shen and Gould, 1991). This suggested to us that the M(r) 94,000 vitamin K-dependent carboxylase may contain a hydroquinone epoxidase domain discrete from other regions of the protein responsible for carboxylation, recognition of the -carboxylation recognition site (-CRS) in the propeptides of vitamin K-dependent proteins (Jorgensen et al., 1987), and membrane association. While no sequence data are available regarding the purified microbial hydroquinone epoxidases, our strategy to test this hypothesis was to pursue a mutagenesis-based structure-function analysis of the carboxylase.

The recent affinity purification of bovine liver carboxylase (Wu et al., 1991b; Kuliopulos et al., 1992a) has lead to cloning the full-length human liver cDNA (Wu et al., 1991a) and the full-length bovine liver cDNA (Rehemtulla et al., 1993). While expression of these cDNAs in heterologous mammalian cells results in an increase in carboxylase activity above endogenous background levels (Wu et al., 1991a; Rehemtulla et al., 1993), the expression of the bovine liver cDNA in baculovirus-infected insect cells has been particularly useful due to the lack of endogenous vitamin K-dependent carboxylase activity in these cells (Roth et al., 1993). The carboxylase active site (Kuliopulos et al., 1994) and the -CRS (Yamada et al., 1995) have recently been localized by affinity labeling studies to the amino-terminal third of the vitamin K-dependent carboxylase. In the current study, site-directed mutagenesis was pursued to create a series of mutant enzymes with progressively larger deletions of the carboxyl terminus. Characterization of these mutant proteins, expressed in baculovirus-infected insect cells, provides the first functional evidence to suggest a role for the carboxyl terminus in mediating an interaction with vitamin K hydroquinone.


MATERIALS AND METHODS

Chemicals

FLEEL, CHAPS, sodium borohydride, and L-alpha-phosphatidylcholine (Type V-E) were from Sigma. Vitamin K(1) (10 mg/ml, Aquamephyton) was from Merck Sharp and Dohme. ProPT18, a synthetic peptide corresponding to residues -18 to -1 of human proprothrombin, was synthesized and purified as described previously (Ulrich et al., 1988; Hubbard et al., 1989).

Production of Mutant Carboxylase Enzymes

A TAA stop codon was inserted in frame with the coding sequence of the bovine liver carboxylase to produce cDNAs encoding mutant enzymes which terminate prematurely after residues Pro, Pro, and Thr: Cbx 711, Cbx 676, and Cbx 572, respectively. Oligonucleotide-directed in vitro mutagenesis was performed with the Altered Sites Mutagenesis System (Promega) as recommended by the manufacturer. The cDNA encoding bovine liver carboxylase was subcloned from the baculovirus transfer vector pBLII/bCbx (Roth et al., 1993) into the plasmid pALTER-1 (Promega) to create pALTER/Cbx. The plasmid pBLII/bCbx was digested with NheI and HindIII, and the fragment containing the carboxylase coding sequence was directionally subcloned into the isolated mutagenesis phagemid vector pALTER-1 which had been linearized with XbaI and HindIII. The plasmid pALTER/Cbx was used to prepare phagemid single-stranded DNA with the helper bacteriophage R408. Single-stranded ALTER/bCbx phagemid DNA was used as a template in mutagenesis reactions with the following mutagenic oligonucleotides: Cbx711 = 5`-GGCCGCCCTTAAGTGGAGCAGCTGGCCC-3`; Cbx676 = 5`-CGAAATGCCCCTTAAGACGAGCGACTTGTCCGC-3`; and Cbx572 = 5`-CAGAAGAACCAGACTTAAGAGGAGGGAGAAAAAATG-3`.

Mutant plasmids were propagated in the repair minus Escherichia coli strain BMH 71-18 mut S. The rapid identification of mutant plasmids by restriction enzyme analysis with AflII was facilitated by inclusion of an in-frame TAA termination codon (indicated in bold face type) in the context of an AflII restriction site (underlined), which was unique in the carboxylase coding sequence. The mutations were introduced into the baculovirus transfer vector pHis(6)bCbx (Kuliopulos et al., 1994) by replacing a StuI-HindIII fragment containing a portion of the wild-type carboxylase coding sequence with the corresponding StuI-HindIII fragment that was isolated from each mutant plasmid, to create pHis(6)Cbx711, pHis(6)Cbx676, and pHis(6)Cbx572. The presence of each mutation was confirmed by DNA sequence analysis.

Expression of Wild-type and Mutant His(6)-Carboxylase Enzymes

Each baculovirus transfer vector was used to generate a plaque-purified strain of recombinant baculovirus, vHis(6)Cbx711, vHis(6)Cbx676, and vHis(6)Cbx572, respectively (Roth et al., 1993). Wild-type carboxylase and each mutant carboxylase was expressed in Spodoptera frugiperda (Sf9) cells in 50-100 ml suspension cultures, the cells harvested, and microsomes prepared as described previously (Roth et al., 1993). In brief, microsomal pellets (500-750 µl) were resuspended with 2-3 volumes of 25 mM MOPS pH 7.4, 1 M NaCl, 10% (v/v) glycerol, and protease inhibitors (phenylmethylsulfonyl fluoride (100 µg/ml), leupeptin (0.5 µg/ml), pepstatin A (1.0 µg/ml), and aprotinin (2.0 µg/ml)) by sonication with a microtip ultrasonic probe (Heat Systems-Ultrasonic, model W-220) at 4 °C. The recombinant carboxylase was then solubilized by mixing the resuspended microsomes with an equal volume of 1.5% CHAPS, 1.5% phosphatidylcholine, 25 mM MOPS pH 7.4, 1 M NaCl, containing protease inhibitors as above, and sonicating again with ten 5-s pulses. Insoluble material was pelleted by ultracentrifugation at 100,000 times g for 60 min at 4 °C, and the soluble supernatant aliquoted and stored at -80 °C. The soluble enzyme preparations were stable to multiple freeze/thaw cycles provided the glycerol concentration was at least 5% (v/v).

Carboxylase Activity and Kinetic Analyses

The in vitro carboxylase activity assay was performed as described previously (Roth et al., 1993). ^14CO(2) incorporated into the peptide substrate FLEEL was determined in 125 µl final reaction volumes for 30 min at 25 °C (Suttie et al., 1979). Unless stated otherwise, reactions contained 25 mM MOPS pH 7.4, 0.5 M NaCl, 0.16% CHAPS, 0.16% phosphatidylcholine, 8 mM DTT, 0.8 M ammonium sulfate, 16 µM proPT18, 3.6 mM FLEEL, 888 µM vitamin K hydroquinone, and 10 µCi of NaH^14CO(3) (55 mCi/mmol, Amersham Corp.). Vitamin K(1) was reduced in situ with NaBH(4) as described (Kutkow et al., 1993). Assays performed in duplicate included between 0.23 and 6.4 pmol of enyzme/125 µl of reaction. Background counts/minute for assays lacking enzyme, FLEEL, or vitamin K hydroquinone, were 200 to 400 cpm. Background cpm were subtracted from all data points. Determination of kinetic parameters for FLEEL was performed by aliquoting the appropriate quantity of FLEEL into reaction tubes. A master mixture was prepared within 10 min of initiating the assay. This mixture, containing all of the other reaction components including diluted enzyme, was added to the FLEEL to initiate the reaction. Kinetic analyses with vitamin K hydroquinone were performed by aliquoting the vitamin K hydroquinone into tubes already containing DTT, such that the final concentration of DTT after addition of the master mixture was 8 mM. When necessary, vitamin K hydroquinone was diluted in N(2)-saturated ethanol immediately prior to use. The master mixture contained all of the reaction components except DTT and vitamin K hydroquinone. Enzyme was added to the mixture immediately prior to use, and the reactions were initiated by addition of the enzyme-containing master mixture. Estimates of K(m) and V(max) were made using the non-linear least-squares regression analysis of the Michaelis-Menten equation in Sigma Plot (Jandel Scientific).

Vitamin K Hydroquinone Epoxidase Activity Assay

Reactions were performed exactly as described for carboxylase assays, except that NaHCO(3) was substituted for NaH^14CO(3). Vitamin K 2,3-epoxide was determined as described previously (Haroon et al., 1986; Sadowski et al., 1988) At the completion of a 30-min incubation at 25 °C in sealed tubes, the vitamin K contained in a 125-µl reaction volume was extracted with 2 volumes of ethanol and 3 volumes of hexane. The sample was centrifuged for 5 min at 1000 times g, the hexane phase transferred to a tube, and the solvent evaporated to dryness. The pellet was resuspended in methanol, and a portion of the sample was injected onto an analytical C(18) Hypersil ODS reverse-phase high performance liquid chromatography column (4.6 mm times 250 mm, 5 µM). The column was eluted at a flow rate of 1 ml/min with 10% dichloromethane, 90% methanol (v/v) which was saturated with nitrogen. Vitamin K 2,3-epoxide was detected by absorption at 226 nm and quantitated using a purified standard as a control. All assays were performed in triplicate.

Quantitative Western Blot Analysis with His(6)-T7-beta-galactosidase

His(6)-T7-beta-galactosidase was expressed using the bacterial expression plasmid pET28b in BL21 (DE3) E. coli (Induction Control E, Novagen) as recommended by the manufacturer. Cells were harvested 2 h following induction with isopropylthio-beta-galactoside and stored as frozen pellets at -80 °C until needed. The His(6)-T7-beta-galactosidase fusion protein was purified to homogeneity by immobilized metal-ion affinity chromatography (Porath, 1992) using iminodiacetic acid-Sepharose charged with NiCl(2) (Probond, Invitrogen). The cells from 100 ml of culture were thawed in binding buffer (5 mM imidazole, pH 7.4, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9 with protease inhibitors (phenylmethylsulfonyl fluoride (100 µg/ml), leupeptin (0.5 µg/ml), pepstatin A (1.0 µg/ml), and aprotinin (2.0 µg/ml)). Cells were disrupted by sonication and insoluble material was removed by centrifugation at 10,000 times g for 15 min. The supernatant was applied to 2 ml of resin, washed with 20 column volumes of binding buffer followed by 20 column volumes of 60 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9, and eluted in 4 ml of 200 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9. The protein eluate was adjusted to 50% glycerol, 2.5% beta-mercaptoethanol. Protein concentration was determined with a Bradford Protein assay (Bio-Rad) and a BCA protein assay (Pierce) using bovine serum albumin as a standard. Western blot analysis was performed as described previously (Kuliopulos et al., 1994) using the T7-Tag mouse monoclonal antibody (Novagen). Densitometric scanning of Western blots was performed with an LKB UltraScan laser densitometer.


RESULTS

Expression and Quantitation of Recombinant Wild-type and Mutant Vitamin K-dependent Carboxylase

Site-directed mutagenesis was used to introduce a TAA stop codon at various positions in the 3` end of the cDNA for His(6)-carboxylase, to produce a series of mutant enzymes with progressively larger carboxyl-terminal deletions. The His(6)-carboxylase fusion protein contains a bacteriophage T7 gene 10 leader peptide sequence (His(6)-carboxylase residues -23 to -13) recognized by the T7-Tag mouse monoclonal antibody (Kuliopulos et al., 1994). This antigenic epitope lies outside the coding region of the carboxylase, is present in all of the mutant proteins expressed, and facilitates Western blot analyses to accurately determine recombinant protein levels in microsomal preparations. The recombinant His(6)-carboxylase fusion protein has the same kinetic properties as the wild-type enzyme from bovine liver lacking the NH(2)-terminal His(6) tag (Kuliopulos et al., 1994), making it useful for mutagenesis-based structure-function studies.

Functional characterization of the wild-type and mutant enzymes were performed with partially purified solubilized microsomal protein preparations from baculovirus-infected Sf9 cells. The enzyme was stable in these preparations in contrast to more highly purified preparations. CHAPS-solubilized total microsomal protein was analyzed by SDS-PAGE in 10% gels under reducing conditions, followed by Western blotting with the T7-Tag antibody (Fig. 1). The wild-type carboxylase migrated at a molecular mass of 94 kDa in agreement with the previous estimation of 90 kDa for the carboxylase expressed in Sf9 cells (Roth et al., 1993) plus 4 kDa for the 34 amino acid His(6)-T7-containing amino-terminal extension. The observed mass of Cbx 711, Cbx 676, and Cbx 572 was 88, 84, and 71 kDa, respectively. These results are in good agreement with the predicted mass of 86.3, 81.9, and 70.1 kDa, respectively. Cbx 572 preparations demonstrated minor quantities of two other T7 containing proteins: a smaller protein most likely corresponding to a proteolytic fragment and a larger species, possibly corresponding to a dimer or an aggregate of the recombinant protein. The predominant band detected in the lane corresponded to the expected size of the mutant protein terminating at residue 572 and represented about 90% of the total protein in the lane detected by the T7 antibody. Wild-type carboxylase, Cbx 711, and Cbx 676 yielded single T7 antibody-reactive bands.


Figure 1: Western blot analysis of recombinant wild-type and mutant vitamin K-dependent carboxylases expressed in Sf9 cells. Solubilized microsomal protein from infected Sf9 cells, containing between 50 and 100 fmol of enzyme, was subjected to SDS-PAGE under reducing conditions in 10% gels. The recombinant proteins in each sample were detected with the T7-Tag monoclonal antibody. Lane 1, wild-type carboxylase; lane 2, Cbx 711; lane 3, Cbx 676; lane 4, Cbx 572. Molecular size standards are shown on the left.



The concentration of each preparation of wild-type and mutant carboxylase enzyme used in kinetic analyses was determined by Western blot analysis with T7 antibody employing as a standard, purified His(6)-T7-beta-galactosidase, which was homogeneous by SDS-PAGE analysis. His(6)-T7-beta-galactosidase contains the same T7-Tag antigenic epitope present in the wild-type and mutant carboxylase proteins. Varying known concentrations of His(6)-T7-beta-galactosidase were included on the same gel as the carboxylase preparations. Quantitation of the His(6)-T7-beta-galactosidase by laser densitometric scanning of a Western blot developed with the T7 antibody yielded a linear relationship between the quantity of His(6)-T7-beta-galactosidase, from 2 to 40 ng of protein/lane, and the area within the densitometer peak (data not shown). Quantitation for the T7-containing carboxylase species could be obtained in the femtomolar range.

Determination of K(m) and V(max) for Carboxylation of FLEEL

The wild-type carboxylase displayed Michaelis-Menten kinetic behavior with concentrations of FLEEL up to 10 mM. Above 10 mM substrate, apparent substrate inhibition was observed for His(6)-carboxylase, as previously observed for the purified bovine liver carboxylase (Morris et al., 1993). Thus, FLEEL concentrations below 10 mM were used to derive all kinetic parameters. The K(m) of the wild-type enzyme for FLEEL was 0.87 mM ( Table 1and Fig. 2A); this value is similar to the previously reported value of 1 mM for the purified bovine liver derived enzyme (Morris et al., 1993). Cbx 711 and Cbx 676 exhibited similar K(m) values to wild-type carboxylase ( Table 1and Fig. 2, B and C). No carboxylase activity was observed for Cbx 572 under the assay conditions employed. Cbx 572 had no measurable activity when tested with FLEEL concentrations as high as 30 mM.




Figure 2: Dependence of carboxylase activity on the concentration of FLEEL. CHAPS-solubilized total microsomal protein from baculovirus-infected Sf9 cells was used as a source for each recombinant enzyme tested. With the concentrations of FLEEL used, the enzymes displayed Michaelis-Menten behavior toward the glutamate containing peptide substrate FLEEL. To compare the V(max) of each sample, the measured V(max) was normalized for the quantity of enzyme used in each individual assay. Plots: A, wild-type carboxylase; B, Cbx 711; C, Cbx 676. The absolute activities measured for each mutant reached approximately 10,000 cpm/30 min assay which was significantly above background determinations for each assay (200-400 cpm). The data represent the average of duplicate points for each concentration of FLEEL.



The V(max) of each enzyme species for the substrate FLEEL was determined ( Table 1and Fig. 2, A-C). While the V(max) for wild-type carboxylase and Cbx 711 were similar, a 28-fold decrease was noted for Cbx 676 compared to the wild-type enzyme. The V(max)/K(m) values were similar for the wild-type and Cbx 711 enzymes; however, a significantly lower value (about 15-fold) was obtained for Cbx 676 (Table 1), demonstrating that Cbx 676 is much less efficient at catalyzing the carboxylation of FLEEL compared to wild-type enzyme or Cbx 711.

Propeptide Stimulation of Carboxylation of FLEEL

The carboxylase binds to conserved sequences in the propeptides of the vitamin K-dependent proteins (Jorgensen et al., 1987; Ulrich et al., 1988; Huber et al., 1990). The inclusion of synthetic peptides corresponding to these highly conserved propeptide sequences has been shown to stimulate the carboxylation of small peptide substrates (Knobloch and Suttie, 1987; Ulrich et al., 1988). This enhancement is reported to be the result of an approximately 5-10-fold reduction in the K(m) for FLEEL (Knobloch and Suttie, 1987; Cheung et al., 1989). The in vitro vitamin K-dependent carboxylation of FLEEL by the recombinant carboxylase expressed in insect cells is also stimulated by an 18-residue peptide corresponding to the propeptide of factor IX or prothrombin (Roth et al., 1993). We investigated whether the diminished V(max) observed with Cbx 676 was due to a loss of responsiveness to proPT18, which is a component of our standard carboxylase assay. As shown in Table 2, inclusion of 8 or 16 µM propeptide maximally stimulated carboxylation of FLEEL about 2.5-fold for each of the enzyme species. Cbx 572 had no measurable activity when tested with propeptide concentrations as high as 200 µM. Although no conclusion can be drawn for Cbx 572, these data suggest that deletions in Cbx 711 and Cbx 676 have not influenced the enzyme-propeptide interaction.



Determination of K(m) and V(max) for Vitamin K Hydroquinone

Wild-type carboxylase was determined to have a K(m) for vitamin K hydroquinone (vitamin KH(2)) of 23.2 µM (Table 3, Fig. 3A), a value which agrees with the reported value of 36 µM for the purified bovine liver enzyme (Morris et al., 1993). At high concentrations of vitamin K hydroquinone (generally greater than 1 mM), inhibition of carboxylase activity was observed. This phenomenon was described previously with the purified bovine liver enzyme and may relate to the vitamin directly, or to other components present in the preparation of vitamin K hydroquinone (Morris et al., 1993). Vitamin K hydroquinone concentrations below those which resulted in activity inhibition were used in experiments that yielded all kinetic parameters. The K(m) of Cbx 711 for vitamin KH(2) was not significantly different from wild-type carboxylase (Table 3, Fig. 3B). However, the K(m) for vitamin KH(2) determined with Cbx 676 was 542 µM, about 23-fold higher than the K(m) determined with wild-type carboxylase (Table 3, Fig. 3C), in contrast to the equivalent K(m) for FLEEL. The V(max) for wild-type carboxylase and Cbx 711 were similar. The V(max) determined for Cbx 676 was approximately 17-fold lower than the V(max) determined for wild-type carboxylase (Table 3, Fig. 3, A-C), comparable with the measured decrease in the Cbx 676 V(max) for FLEEL. The V(max)/K(m) for KH(2) with Cbx 676 was thus reduced about 400-fold compared with wild-type carboxylase. The enzyme Cbx 572 had no measurable activity with vitamin K hydroquinone levels as high as 3.5 mM.




Figure 3: Dependence of carboxylase activity on the concentration of vitamin K hydroquinone. CHAPS-solubilized total microsomal protein from baculovirus-infected Sf9 cells was used as a source of recombinant carboxylase. The enzymes displayed Michaelis-Menten behavior toward the cofactor vitamin K hydroquinone within the concentration ranges used for the assay. To compare the V(max) of each sample, the measured V(max) was normalized for the quantity of enzyme used in each individual assay. Plots: A, wild-type carboxylase; B, Cbx 711; C, Cbx 676. The absolute activities measured for each mutant reached between 10,000 and 20,000 cpm/30 min assay which was significantly above background determinations for each assay (200-400 cpm). The data represent the average of duplicate points for each concentration of vitamin K hydroquinone.



Vitamin K Epoxidase Activity

The amount of vitamin K 2,3-epoxide formed during a standard 30-min carboxylase assay under saturating concentrations of NaHCO(3) was quantitated (Table 4). To facilitate direct comparison for each enzyme form tested, the specific activity of each enzyme in terms of its function as an epoxidase was measured. There was a small (20%) decrease in the amount of vitamin K epoxide generated by Cbx 711 compared to wild-type carboxylase. However, a significant 10-fold decrease in the amount of vitamin K epoxide was observed with Cbx 676. When vitamin K epoxide was substituted for vitamin K hydroquinone as a cofactor in the in vitro carboxylase assay, there was no measurable incorporation of ^14CO(2) into FLEEL, suggesting that there was no vitamin K epoxide reductase activity present in the Sf9 microsomal protein preparation capable of recycling vitamin K epoxide to vitamin K hydroquinone. Comparison of ^14CO(2) incorporation into FLEEL with vitamin K epoxide formation, assuming dilution of ^14CO(2) specific radioactivity by endogenous CO(2)/HCO(3) of about 2 mM (Wood and Suttie, 1988), revealed that carboxylation and epoxidation were coupled under the conditions employed for this assay.




DISCUSSION

The vitamin K-dependent -carboxylase is a unique microsomal enzyme that catalyzes both the conversion of vitamin K hydroquinone to vitamin K epoxide and the generation of -carboxyglutamic acid from glutamic acid residues on substrate proteins. The precise role of vitamin K and the mechanism of action of this enyzme is not known. However, studies of partially purified and purified carboxylase have shown that the carboxylase and epoxidase activities are associated with the same protein. Nonetheless, at low concentrations of glutamyl-containing substrates or CO(2), this enzyme exhibits epoxidase activity without concomitant carboxylase activity. This uncoupling of epoxidation and carboxylation suggests a complex enzyme mechanism for the -carboxylase. The current study explores the relationship of the epoxidase and the carboxylase activities of COOH-terminal truncation mutants of the carboxylase.

Site-directed mutagenesis of the carboxylase cDNA was used to produce a series of progressively larger carboxyl-terminal deletions. Western blot analysis of each expressed protein confirmed that the insertion of a TAA stop codon resulted in the synthesis of a truncated protein. The T7-tag antigenic epitope was located at the extreme amino terminus of each recombinant protein, further substantiating that the differences in size were due to loss of variable portions of the carboxyl terminus. Mutagenesis of the carboxylase carboxyl terminus did not alter the amino-terminal T7 epitope. We assumed that each of the four different carboxylase proteins was recognized by the T7-tag monoclonal antibody with equivalent affinity, facilitating quantitation by Western blot analysis. The purification of each mutant by virtue of the His(6) tag with immobilized metal-ion affinity chromatography under native conditions proved to be inefficient and enzyme activity was highly unstable. Our previously reported purification of the enzyme under denaturing conditions was not useful for structure-function analyses (Kuliopulos et al., 1994). The propeptide affinity chromatography employed for native enzyme from bovine liver produced only very low yields of pure active enzyme (Kuliopulos et al., 1992a), and the possible disruption of the carboxylasepropeptide interaction by mutagenesis precluded this technique for the purification of mutant enyzmes. To circumvent these problems, functional characterization was performed with crude solubilized microsomal protein, where the enzyme activity proved to be very stable. The characterization of purified bovine liver carboxylase demonstrated similar characteristics to carboxylase activity in crude solubilized liver microsomes, suggesting that we could anticipate similar results with each mutant when analyzed in crude solubilized microsome preparations (Morris et al., 1993).

The K(m) values for FLEEL with wild-type carboxylase, Cbx 711, and Cbx 676 were the same. The lack of effect of missing portions of the carboxyl terminus on FLEEL recognition is consistent with previous studies that localize the site of attachment of N-bromoacetyl-FLEELY, an inactivating substrate, to the amino terminus, in the first 218 residues of the 758 residue carboxylase (Kuliopulos et al., 1994). The synthetic peptide ProPT18, corresponding to residues -18 to -1 of human prothrombin, was able to stimulate the carboxylation of FLEEL, in our in vitro assay system. The wild-type carboxylase, Cbx 711, and Cbx 676 demonstrated similar activation by the propeptide. These findings are consistent with recent photoactivatable cross-linking studies which localized the factor IX propeptide-binding site also to the amino-terminal third of the enzyme (Yamada et al., 1995). We conclude that removal of up to the carboxyl-terminal 82 residues of the enzyme does not significantly influence the ability of the enzyme to interact with glutamate residues contained in peptide substrates or the -CRS contained in the propeptide of prothrombin.

When the K(m) for vitamin K hydroquinone was determined for wild-type carboxylase, Cbx 711, and Cbx 676, wild-type carboxylase and Cbx 711 demonstrated a similar K(m), while that of Cbx 676 was clearly different. The K(m) was significantly increased (23-fold), and the V(max) and V(max)/K(m) were significantly reduced (17- and 400-fold, respectively), revealing that Cbx 676 has decreased catalytic efficiency toward vitamin K hydroquinone compared to the wild-type carboxylase and Cbx 711. The analysis of vitamin K 2,3-epoxide formed during the in vitro carboxylation reaction corroborates that Cbx 676 has a low specific epoxidase activity. These data support the conclusion that deletion of the 47 most carboxyl-terminal residues (Cbx 711) from the carboxylase has no significant effect on the enzyme interaction with vitamin K hydroquinone, while deletion of an additional 35 carboxyl-terminal residues (Cbx 676) selectively influences the interaction of carboxylase with vitamin K hydroquinone. These findings agree with the concept that vitamin K 2,3-epoxide formation and -glutamyl carboxylation, while normally coupled, are separate processes, with the latter being dependent on the former. Under limiting concentrations of glutamyl peptide substrate or bicarbonate, the formation of vitamin K epoxide can occur without carboxylation. The basicity enhancement mechanism proposed by Ham and Dowd(1990) helps to explain this phenomenon. In that formulation, the enzyme first acts as a dioxygenase with vitamin K hydroquinone as a substrate, by way of an unstable dioxetane intermediate that rearranges to form a vitamin K alkoxide base which can abstract the -glutamyl hydrogen from a glutamic acid residue. The neutralized vitamin K alkoxide can then dehydrate to form vitamin K 2,3-epoxide and H(2)O, and enzyme-mediated capture of CO(2) by the carbanion at the -carbon of glutamate can proceed.

The complete loss of activity demonstrated with Cbx 572 may be secondary to deletion of regions required for vitamin K recognition and/or epoxidase activity, resulting in the subsequent loss of carboxylase activity. However, it is also possible that this deletion has resulted in aberrant protein folding which destabilizes the enzyme structure, leading to loss of function on that basis. Evidence which suggests this possibility relates to the appearance of a probable proteolytic fragment and aggregate seen only in preparations of this enzyme. Therefore, we are unable at present to draw conclusions from the lack of activity of Cbx 572.

The altered interaction of Cbx 676 with vitamin K hydroquinone may suggest that a vitamin K epoxidase activity domain may reside in the COOH terminus or be influenced by residues located in the COOH terminus of the carboxylase. The hydrophobic region from residues 684 to 710 which was deleted in this construct may present an important hydrophobic surface which promotes an interaction with vitamin K hydroquinone. Two microbial hydroquinone epoxidases which catalyze the formation of epoxysemiquinone antibiotics similar in structure to vitamin K 2,3-epoxide were recently purified and characterized (Shen and Gould, 1991), although no sequence data are yet available. These soluble enzymes may share a similar mechanism of epoxidase activity with the carboxylase, given the structural similarities of their substrates, and their small subunit size suggests that epoxidase activity may reside in a functional domain of the carboxylase. The recent structural determination of soybean lipoxygenase-1 solved to 2.6-Å resolution is also of interest (Boyington et al., 1993). This lipoxygenase shares 19.8% sequence identity with the carboxylase over 91 overlapping amino acids in addition to 50% conservative substitutions over the same range (carboxylase residues 467-548) (Pearson and Lipman, 1988). Lipoxygenases contain a non-heme iron and utilize molecular oxygen in the dioxygenation of arachidonic acid. The carboxylase acts as a dioxygenase on its cofactor, vitamin K hydroquinone; however, the mechanistic similarity between these enzymes is presently unknown. The structure of soybean lipoxygenase-1 reveals two domains, a 146-residue beta barrel which makes only loose contact with a 693-residue helical bundle (Boyington et al., 1993). The carboxyl-terminal helical bundle domain, which is conserved in both plant and mammalian lipoxygenases, coordinates iron and contains the dioxygenase activity of the lipoxygenase, and it is this domain that shares sequence similarity with the carboxylase. Whether a discrete epoxidase domain exists in the carboxyl terminus of the carboxylase, separate from the carboxylase domain, or whether epoxidase activity is influenced by this region is beyond the scope of this paper to determine. However, investigations to further test this hypothesis are under way with more refined deletion and substitution mutations in the carboxyl terminus, as well as near the active site and propeptide binding site, localized to the amino terminus in previous investigations (Kuliopulos et al., 1994; Yamada et al., 1995).


FOOTNOTES

*
This work was supported by Grant HL42443 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of Physician Scientist Award HL02574 from the National Institutes of Health.

(^1)
The abbreviations used are: Gla, -carboxyglutamic acid; carboxylase, vitamin K-dependent -glutamyl carboxylase; Cbx, carboxylase; -CRS, -carboxylase recognition site; vitamin KH(2), vitamin K hydroquinone; vitamin KO, vitamin K 2,3-epoxide; FLEEL, Phe-Leu-Glu-Glu-Leu; proPT18, synthetic peptide corresponding to human proprothrombin residues -18 to -1 (His-Val-Phe-Leu-Ala-Pro-Gln-Gln-Ala-Arg-Ser-Leu-Leu-Gln-Arg-Val-Arg-Arg); Sf9, Spodoptera frugiperda; DTT, dithiothreitol; MOPS, 3-(n-morpholino)propanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Jim Sadowski and Ken Davidson for providing purified vitamin K 2,3-epoxide as a standard and for valuable advice regarding HPLC analysis of vitamin K epoxide.


REFERENCES

  1. Boyington, J. C., Gaffney, B. J. & Amzel, L. M. (1993) Science 260, 1482-1486 [Medline] [Order article via Infotrieve]
  2. Cheung, A., Engelke, J. A., Sanders, C. & Suttie, J. W. (1989) Arch. Biochem. Biophysics 274, 574-581 [Medline] [Order article via Infotrieve]
  3. Dowd, P., Ham, S. & Hershline, R. (1992) J. Am. Chem. Soc. 114, 7613-7617
  4. Furie, B. & Furie, B. C. (1988) Cell 53, 505-518 [Medline] [Order article via Infotrieve]
  5. Gould, S. J. & Shen, B. (1991) J. Am. Chem. Soc. 113, 684-686
  6. Ham, S. W. & Dowd, P. (1990) J. Am. Chem. Soc. 112, 1660-1661
  7. Haroon, Y., Bacon, D. S. & Sadowski, J. A. (1986) Clin. Chem. 32, 1925-1929 [Abstract/Free Full Text]
  8. Hubbard, B. R., Ulrich, M. M. W., Jacobs, M., Vermeer, C., Walsh, C., Furie, B. & Furie, B. C. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 6893-6897 [Abstract]
  9. Huber, P., Schmitz, T., Griffin, J., Jacobs, M., Walsh, C., Furie, B. & Furie, B. C. (1990) J. Biol. Chem. 265, 12467-12473 [Abstract/Free Full Text]
  10. Jorgensen, M. J., Cantor, A. B., Furie, B. C., Brown, C. L., Shoemaker, C. B. & Furie, B. (1987) Cell 48, 185-191 [Medline] [Order article via Infotrieve]
  11. Knobloch, J. E. & Suttie, J. W. (1987) J. Biol. Chem. 262, 15334-15337 [Abstract/Free Full Text]
  12. Kuliopulos, A., Cieurzo, C. E., Furie, B., Furie, B. C. & Walsh, C. T. (1992a) Biochemistry 31, 9436-9444 [Medline] [Order article via Infotrieve]
  13. Kuliopulos, A., Hubbard, B. R., Lam, Z., Koski, I. J., Furie, B., Furie, B. C. & Walsh, C. T. (1992b) Biochemistry 31, 7722-7728 [Medline] [Order article via Infotrieve]
  14. Kuliopulos, A., Nelson, N. P., Yamada, M., Walsh, C. T., Furie, B., Furie, B. C. & Roth, D. A. (1994) J. Biol. Chem. 269, 21364-21370 [Abstract/Free Full Text]
  15. Kutkow, K. J., Roth, D. A., Porter, T. J., Furie, B. C. & Furie, B. (1993) Methods Enzymol. 222, 435-449 [Medline] [Order article via Infotrieve]
  16. Larson, A. E., Friedman, P. A. & Suttie, J. W. (1981) J. Biol. Chem. 256, 11032-11035 [Abstract/Free Full Text]
  17. Morris, D. P., Soute, B. A. M., Vermeer, C. & Stafford, D. W. (1993) J. Biol. Chem. 268, 8735-8742 [Abstract/Free Full Text]
  18. Pearson, W. R. & Lipman, D. J. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 2444-2448 [Abstract]
  19. Porath, J. (1992) Protein Exp. Purification 3, 263-281 [Medline] [Order article via Infotrieve]
  20. Rehemtulla, A., Roth, D. A., Wasley, L. C., Kuliopulos, A., Walsh, C. T., Furie, B., Furie, B. C. & Kaufman, R. J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4611-4615 [Abstract]
  21. Roth, D. A., Rehemtulla, A., Kaufman, R. J., Walsh, C. T., Furie, B. & Furie, B. C. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8372-8376 [Abstract/Free Full Text]
  22. Shen, B. & Gould, S. J. (1991) Biochemistry 30, 8936-8944 [Medline] [Order article via Infotrieve]
  23. Soriano-Garcia, M., Padmanabhan, K., de Vos, A. M. & Tulinsky, A. (1992) Biochemistry 31, 2554-2566 [Medline] [Order article via Infotrieve]
  24. Suttie, J. W. (1985) Annu. Rev. Biochem. 54, 459-477 [CrossRef][Medline] [Order article via Infotrieve]
  25. Suttie, J. W., Lehrman, S. R., Geweke, L. O., Hageman, J. M. & Rich, D. H. (1979) Biochem. Biophys. Res. Commun. 86, 500-507 [Medline] [Order article via Infotrieve]
  26. Ulrich, M. M. W., Furie, B., Jacobs, M. R., Vermeer, C. & Furie, B. C. (1988) J. Biol. Chem. 263, 9697-9702 [Abstract/Free Full Text]
  27. Wood, G. M. & Suttie, J. W. (1988) J. Biol. Chem. 263, 3234-3239 [Abstract/Free Full Text]
  28. Wu, S.-M., Cheung, W.-F., Frazier, D. & Stafford, D. W. (1991a) Science 254, 1634-1636 [Medline] [Order article via Infotrieve]
  29. Wu, S.-M., Morris, D. P. & Stafford, D. W. (1991b) Proc. Natl. Acad. Sci. U. S. A. 88, 2236-2240 [Abstract]
  30. Yamada, M., Kuliopulos, A., Nelson, N. P., Roth, D. A., Furie, B., Furie, B. C. & Walsh, C. T. (1995) Biochemistry 34, 481-489 [Medline] [Order article via Infotrieve]

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