(Received for publication, November 17, 1994)
From the
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
terminus.
Vitamin K-dependent -glutamyl carboxylase catalyzes the
post-translational modification of glutamate to
-carboxyglutamate
(Gla) (
)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
O. The mechanism of
carboxylase activity is under intense study. A recently proposed
basicity enhancement model (Ham and Dowd, 1990) supported by
O
-labeling experiments (Dowd et al.,
1992; Kuliopulos et al., 1992b) proposes that CO
fixation is a late-stage reaction-terminating event which follows
O
-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
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
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.
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 pHisbCbx (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
Cbx711,
pHis
Cbx676, and pHis
Cbx572. The presence of
each mutation was confirmed by DNA sequence analysis.
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-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-T7-
-galactosidase, which was homogeneous by
SDS-PAGE analysis. His
-T7-
-galactosidase contains the
same T7-Tag antigenic epitope present in the wild-type and mutant
carboxylase proteins. Varying known concentrations of
His
-T7-
-galactosidase were included on the same gel as
the carboxylase preparations. Quantitation of the
His
-T7-
-galactosidase by laser densitometric scanning
of a Western blot developed with the T7 antibody yielded a linear
relationship between the quantity of
His
-T7-
-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.
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 of each sample, the measured V
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 of each enzyme species for the substrate FLEEL
was determined ( Table 1and Fig. 2, A-C). While
the V
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
/K
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.
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 of each sample, the measured V
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.
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
,
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 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 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 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
, while that of
Cbx 676 was clearly different. The K
was
significantly increased (23-fold), and the V
and V
/K
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
O, and enzyme-mediated
capture of CO
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 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).