Vitamin K-dependent Research Articles

Vitamin K, an example of triage theory: is micronutrient inadequacy linked to diseases of aging?

The triage theory posits that some functions of micronutrients (the ≈40 essential vitamins, minerals, fatty acids, and amino acids) are restricted during shortage and that functions required for short-term survival take precedence over those that are less essential. Insidious changes accumulate as a consequence of restriction, which increases the risk of diseases of aging. For 16 known vitamin K-dependent (VKD) proteins, we evaluated the relative lethality of 11 known mouse knockout mutants to categorize essentiality. Results indicate that 5 VKD proteins that are required for coagulation had critical functions (knockouts were embryonic lethal), whereas the knockouts of 5 less critical VKD proteins [osteocalcin, matrix Gla protein (Mgp), growth arrest specific protein 6, transforming growth factor β-inducible protein (Tgfbi or βig-h3), and periostin] survived at least through weaning. The VKD γ-carboxylation of the 5 essential VKD proteins in the liver and the 5 nonessential proteins in nonhepatic tissues sets up a dichotomy that takes advantage of the preferential distribution of dietary vitamin K1 to the liver to preserve coagulation function when vitamin K1 is limiting. Genetic loss of less critical VKD proteins, dietary vitamin K inadequacy, human polymorphisms or mutations, and vitamin K deficiency induced by chronic anticoagulant (warfarin/coumadin) therapy are all linked to age-associated conditions: bone fragility after estrogen loss (osteocalcin) and arterial calcification linked to cardiovascular disease (Mgp). There is increased spontaneous cancer in Tgfbi mouse knockouts, and knockdown of Tgfbi causes mitotic spindle abnormalities. A triage perspective reinforces recommendations of some experts that much of the population and warfarin/coumadin patients may not receive sufficient vitamin K for optimal function of VKD proteins that are important to maintain long-term health.

Roles for Vitamin K Beyond Coagulation

Recent interest in vitamin K has been motivated by evidence of physiological roles beyond that of coagulation. Vitamin K and vitamin K-dependent (VKD) proteins may be involved in regulation of calcification, energy metabolism, and inflammation. However, the evidence for many of these proposed roles in the maintenance of health is equivocal. There is also an emerging viewpoint that the biochemical function of vitamin K may extend beyond that of a cofactor for the VKD carboxylation of glutamyl residues (Glus) to carboxylated Glus in VKD proteins.

Insight into the Coupling Mechanism of the Vitamin K-Dependent Carboxylase: Mutation of Histidine 160 Disrupts Glutamic Acid Carbanion Formation and Efficient Coupling of Vitamin K Epoxidation to Glutamic Acid Carboxylation

Vitamin K-dependent (VKD) proteins become activated by the VKD carboxylase, which converts Glu's to carboxylated Glu's (Gla's) in their Gla domains. The carboxylase uses vitamin K epoxidation to drive Glu carboxylation, and the two half-reactions are coupled in 1:1 stoichiometry by an unknown mechanism. We now report the first identification of a residue, His160, required for coupling. A H160A mutant showed wild-type levels of epoxidation but substantially less carboxylation. Monitoring proton abstraction using a peptide with Glu tritiated at the gamma-carbon position revealed that poor coupling was due to impaired carbanion formation. H160A showed a 10-fold lower ratio of tritium release to vitamin K epoxidation than wild-type enzyme (i.e., 0.12 versus 1.14, respectively), which could fully account for the fold decrease in coupling efficiency. The Ala substitution in His160 did not affect the K m for vitamin K and caused only a 2-fold increase in the K m for Glu and 2-fold decrease in the activation of vitamin K epoxidation by Glu. The H160A K m for CO 2 was 5-fold higher than the wild-type enzyme. However, the k cat for H160A carboxylation was 8-9-fold lower than the wild-type enzyme with all three substrates (i.e., Glu, CO 2, and vitamin K), suggesting a catalytic role for His160 in carbanion formation. We propose that His160 facilitates the formation of the transition state for carbanion formation. His160 is highly conserved in metazoan VKD carboxylases but not in some bacterial orthologues (acquired by horizontal gene transfer), which has implications for how bacteria have adapted the carboxylase for novel functions.

Brønsted Analysis Reveals Lys218 as the Carboxylase Active Site Base That Deprotonates Vitamin K Hydroquinone To Initiate Vitamin K-Dependent Protein Carboxylation

The vitamin K-dependent (VKD) carboxylase converts Glu's to carboxylated Glu's in VKD proteins to render them functional in a broad range of physiologies. The carboxylase uses vitamin K hydroquinone (KH(2)) epoxidation to drive Glu carboxylation, and one of its critical roles is to provide a catalytic base that deprotonates KH(2) to allow epoxidation. A long-standing model invoked Cys as the catalytic base but was ruled out by activity retention in a mutant where every Cys is substituted by Ala. Inhibitor analysis of the cysteine-less mutant suggested that the base is an activated amine [Rishavy et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 13732-13737], and in the present study, we used an evolutionary approach to identify candidate amines, which revealed His160, His287, His381, and Lys218. When mutational analysis was performed using an expression system lacking endogenous carboxylase, the His to Ala mutants all showed full epoxidase activity but K218A activity was not detectable. The addition of exogenous amines restored K218A activity while having little effect on wild type carboxylase, and pH studies indicated that rescue was dependent upon the basic form of the amine. Importantly, Brønsted analysis that measured the effect of amines with different pK(a) values showed that K218A activity rescue depended upon the basicity of the amine. The combined results provide strong evidence that Lys218 is the essential base that deprotonates KH(2) to initiate the reaction. The identification of this base is an important advance in defining the carboxylase active site and has implications regarding carboxylase membrane topology and the feedback mechanism by which the Glu substrate regulates KH(2) oxygenation.

Compound heterozygosity of novel missense mutations in the gamma-glutamyl-carboxylase gene causes hereditary combined vitamin K–dependent coagulation factor deficiency

AbstractHereditary combined vitamin K–dependent (VKD) coagulation factor deficiency is an autosomal recessive bleeding disorder associated with defects in either the γ-carboxylase, which carboxylates VKD proteins to render them active, or the vitamin K epoxide reductase (VKORC1), which supplies the reduced vitamin K cofactor required for carboxylation. Such deficiencies are rare, and we report the fourth case resulting from mutations in the carboxylase gene, identified in a Tunisian girl who exhibited impaired function in hemostatic VKD factors that was not restored by vitamin K administration. Sequence analysis of the proposita did not identify any mutations in the VKORC1 gene but, remarkably, revealed 3 heterozygous mutations in the carboxylase gene that caused the substitutions Asp31Asn, Trp157Arg, and Thr591Lys. None of these mutations have previously been reported. Family analysis showed that Asp31Asn and Thr591Lys were coallelic and maternally transmitted while Trp157Arg was transmitted by the father, and a genomic screen of 100 healthy individuals ruled out frequent polymorphisms. Mutational analysis indicated wild-type activity for the Asp31Asn carboxylase. In contrast, the respective Trp157Arg and Thr591Lys activities were 8% and 0% that of wild-type carboxylase, and their compound heterozygosity can therefore account for functional VKD factor deficiency. The implications for carboxylase mechanism are discussed.

R-VKORC1 Expression in Factor IX BHK Cells Increases the Extent of Factor IX Carboxylation but Is Limited by Saturation of Another Carboxylation Component or by a Shift in the Rate-Limiting Step

Carboxylation of vitamin K-dependent (VKD) proteins is required for their activity and depends on reduced vitamin K generated by vitamin K oxidoreductase (VKOR) and a redox protein that regenerates VKOR activity. VKD protein carboxylation is inefficient in mammalian cells, and to understand why carboxylation becomes saturated, we developed an approach that directly measures the extent of intracellular VKD protein carboxylation. Analysis of factor IX (fIX)-expressing BHK cells indicated that slow egress of fIX from the endoplasmic reticulum and preferential secretion of the carboxylated form contribute to secreted fIX being more fully carboxylated. The analysis also revealed the first reported in vivo VKD protein turnover, which was 14-fold faster than that which occurs in vitro, suggesting facilitation of this process in vivo. r-VKORC1 expression increased the rate of fIX carboxylation and the extent of secreted carboxylated fIX approximately 2-fold, which shows that carboxylation is the rate-limiting step in fIX turnover and which was surprising because turnover in vitro is limited by release of carboxylated fIX. Interestingly, the increases were significantly smaller than the amount of VKOR overexpression (15-fold). However, when cell extracts were tested in single-turnover experiments in vitro, where redox protein is functionally substituted with dithiothreitol, VKOR overexpression increased the fIX carboxylation rate 14-fold, showing r-VKORC1 is functional for supporting fIX carboxylation. These data indicate that the effect of VKOR overexpression is limited in vivo, possibly because a carboxylation component like the redox protein becomes saturated or because another step is now rate-limiting. The studies illustrate the complexity of carboxylation and potential importance of component stoichiometry to overall efficiency.

Compound Heterozygosity in the Novel Mutations W157R and T591K in the γ-Glutamyl Carboxylase Gene Causes Hereditary Combined Vitamin K-Dependent Coagulation Factor Deficiency in a Tunisian Family.

Combined deficiency in vitamin K-dependent (VKD) coagulation factors is an autosomal recessive bleeding disorder associated with defects in either the VKD carboxylase which converts Glus to Glas in VKD proteins to render them active or the vitamin K epoxide reductase (VKORC1) which supplies the reduced vitamin K cofactor required for carboxylation. Such defects are rare, and we now report the fourth case of deficiency caused by mutations in the carboxylase gene. The mutations were identified in a two year old Tunisian girl who exhibited impaired function in several VKD procoagulant and anticoagulant factors that was not restored by vitamin K administration. Sequence analysis of the propositus did not identify any mutations in the VKORC1 gene but, remarkably, revealed three heterozygous mutations in the carboxylase gene, D31N, W157R and T591K within exons 2, 4 and 13, respectively. None of these mutations have previously been reported. Family analysis showed that D31N and T591K were coallelic and transmitted by the mother while W157R was transmitted by the father. The mutations were not found in the genomes of 200 normal subjects, ruling out frequent polymorphisms. Mutational analysis indicated wild type activity for the D31N carboxylase. In contrast, the W157R and T591K enzymes had activities that were, respectively, 7% and 0% that of wild type carboxylase, and their compound heterozygosity can therefore account for defective carboxylation. Residues 157 and 591 are both highly evolutionarily conserved, and residue 157 lies within a region previously suggested to be important for carboxylase binding to VKD Glus or propeptide. However, the hydrophobic nature of this region and inability of vitamin K administration to restore VKD protein function alternatively suggests that residue 157 may be important for vitamin K binding.

The Vitamin K-dependent Carboxylase Has Been Acquired by Leptospira Pathogens and Shows Altered Activity That Suggests a Role Other than Protein Carboxylation

Leptospirosis is an emerging infectious disease whose pathology includes a hemorrhagic response, and sequencing of the Leptospira interrogans genome revealed an ortholog of the vitamin K-dependent (VKD) carboxylase as one of several hemostatic proteins present in the bacterium. Until now, the VKD carboxylase was known to be present only in the animal kingdom (i.e. metazoans that include mammals, fish, snails, and insects), and this restricted distribution and high sequence similarity between metazoan and Leptospira orthologs strongly suggests that Leptospira acquired the VKD carboxylase by horizontal gene transfer. In metazoans, the VKD carboxylase is bifunctional, acting as an epoxidase that oxygenates vitamin K to a strong base and a carboxylase that uses the base to carboxylate Glu residues in VKD proteins, rendering them active in hemostasis and other physiologies. In contrast, the Leptospira ortholog showed epoxidase but not detectable carboxylase activity and divergence in a region of identity in all known metazoan VKD carboxylases that is important to Glu interaction. Furthermore, although the mammalian carboxylase is regulated so that vitamin K epoxidation does not occur unless Glu substrate is present, the Leptospira VKD epoxidase showed unfettered epoxidation in the absence of Glu substrate. Finally, human VKD protein orthologs were not detected in the L. interrogans genome. The combined data, then, suggest that Leptospira exapted the metazoan VKD carboxylase for some use other than VKD protein carboxylation, such as using the strong vitamin K base to drive a new reaction or to promote oxidative damage or depleting vitamin K to indirectly inhibit host VKD protein carboxylation.

THE VITAMIN K–DEPENDENT CARBOXYLASE

The vitamin K-dependent (VKD) carboxylase uses the oxygenation of vitamin K to convert glutamyl residues (Glus) to carboxylated Glus (Glas) in VKD proteins, rendering them active in a broad range of physiologies that include hemostasis, apoptosis, bone development, arterial calcification, signal transduction, and growth control. The carboxylase has a high-affinity site that selectively binds VKD proteins, usually through their propeptide, and also has a second low-affinity site of VKD protein interaction. Propeptide binding increases carboxylase affinity for the Glu substrate, and the coordinated binding of the VKD propeptide and Glu substrate increases carboxylase affinity for vitamin K and activity, possibly through a mechanism of substrate-assisted catalysis. Tethering of VKD proteins to the carboxylase allows clusters of Glus to be modified to Glas by a processive mechanism that becomes disrupted during warfarin therapy. Warfarin inhibits a vitamin K oxidoreductase that generates the reduced vitamin K cofactor required for continuous carboxylation and causes decreased carboxylase catalysis and increased dissociation of partially carboxylated, inactive VKD proteins. The availability of reduced vitamin K may also control carboxylation in r-VKD protein-expressing cells, where the amounts of reduced vitamin K are sufficient for full carboxylation of low, but not high, expression levels of VKD proteins, and where carboxylation is not improved by overexpression of r-carboxylase. This review discusses these recent advances in understanding the mechanism of carboxylation. Also covered is the identification of functional carboxylase residues, a brief description of the role of VKD proteins in mammalian and lower organisms, and the potential impact of quality control components on carboxylation, which occurs in the endoplasmic reticulum during the secretion of VKD proteins.

A new model for vitamin K-dependent carboxylation: the catalytic base that deprotonates vitamin K hydroquinone is not Cys but an activated amine.

Vitamin K-dependent (VKD) proteins require carboxylation for diverse functions that include hemostasis, apoptosis, and Ca(2+) homeostasis, yet the mechanism of carboxylation is not well understood. Combined biochemical and chemical studies have led to a long-standing model in which a carboxylase Cys catalytic base deprotonates vitamin K hydroquinone (KH(2)), leading to KH(2) oxygenation and Glu carboxylation. We previously identified human carboxylase Cys-99 and Cys-450 as catalytic base candidates: Both were modified by N-ethylmaleimide (NEM) and Ser-substituted mutants retained partial activity, suggesting that the catalytic base is activated for increased basicity. Mutants with Cys-99 or Cys-450 substituted by Ala, which cannot ionize to function as a catalytic base, were therefore analyzed. Both single and double mutants had activity, indicating that Cys-99 and Cys-450 do not deprotonate KH(2). [(14)C]NEM modification of C99A/C450A revealed one additional reactive group; however, Ser-substituted mutants of each of the eight remaining Cys retained substantial activity. To unequivocally test, then, whether any Cys or Cys combination acts as the catalytic base, a mutant with all 10 Cys substituted by Ala was generated. This mutant showed 7% wild-type activity that depended on factor IX coexpression, indicating a VKD protein effect on carboxylase maturation. NEM and diethyl pyrocarbonate inhibition suggested that the catalytic base is an activated His. These results change the paradigm for VKD protein carboxylation. The identity of the catalytic base is critical to understanding carboxylase mechanism and this work will therefore impact both reinterpretation of previous studies and future ones that define how this important enzyme functions.

Protein C separation from human blood plasma derivatives using low cost chromatography.

Protein C (PC) deficiency can cause thrombosis, inhibiting oxygen transport to tissue thus resulting in many complications, including death. Present treatment can cause catastrophic bleeding and other major medical problems. PC treatment has no bleeding or skin necrosis problems because it circulates in the blood as a zymogen and is only activated when and where it is needed. The vitamin K dependent (VKD) proteins are homologous proteins, making the separation of PC from plasma extremely difficult. Immobilized metal affinity chromatography (IMAC) is investigated to separate the VKD proteins to replace immunoaffinity chromatography, because of the high cost of monoclonal antibodies. An IDA-Cu column was found effective for the separation of PC from prothrombin, the most harmful contaminant. For Cohn fraction IV-1 separation, a DEAE column was found an efficient initial step, with about 25-fold PC purity increase. Following this step, an IDA-Cu column could remove many contaminants including prothrombin. The combination of DEAE and IDA-Cu resulted in PC purity increase of about 100-fold.

Analysis of equilibrium adsorption isotherms for human protein C purification by immobilized metal affinity chromatography.

Protein C (PC) is a glycoprotein that plays a pivotal role as an anticoagulant, antithromobotic, and thrombolytic therapeutic in the blood coagulation cascade (Esmon, 1989). It is a member of the vitamin K-dependent (VKD) family also consisting of coagulation protein factors VII, IX, X, proteins S, Z and prothrombin. Human PC is synthesized in the liver as a single chain precursor and circulates in the blood primarily as a two chain zymogen that is activated by proteolytic clevage. Activated PC is a potent serine protease that regulates blood coagulation by deactivating factors Va and VIIIa (a: activated form), thus preventing generation of the enzyme factors Xa and thrombin (Wu and Bruley, 1999).KeywordsDeep Vein ThrombosisImmobilize Metal Affinity ChromatographyEquilibrium Adsorption IsothermBlood Coagulation CascadeMaryland Baltimore CountyThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Carboxylase overexpression effects full carboxylation but poor release and secretion of factor IX: implications for the release of vitamin K-dependent proteins.

Vitamin K-dependent (VKD) proteins are modified by the VKD carboxylase as they transit through the endoplasmic reticulum. In a reaction required for their activity, clusters of Glu's are converted to Gla's, and fully carboxylated VKD proteins are normally secreted. In mammalian cell lines expressing high levels of r-VKD proteins, however, under- and uncarboxylated VKD forms are observed. Overexpression of r-carboxylase does not improve carboxylation, but the lack of effect is not understood, and the intracellular events that occur during VKD protein carboxylation have not been investigated. We analyzed carboxylation in 293- and BHK cell lines expressing r-factor IX (fIX) and endogenous carboxylase or overexpressed r-carboxylase. The fIX secreted from the four cell lines was highly carboxylated, indicating fIX-carboxylase engagement during intracellular trafficking. The r-carboxylase was functional for carboxylation: overexpression resulted in a proportional increase in fIX-carboxylase complexes that yielded full fIX carboxylation. Interestingly, the carboxylated fIX product was not efficiently released from the carboxylase in r-fIX/r-carboxylase cells, resulting in decreased fIX secretion. r-Carboxylase overexpression changed the ratios of intracellular fIX to carboxylase, and we therefore developed an in vitro assay to test whether fIX levels affect release. FIX-carboxylase complexes were in vitro carboxylated with or without excess VKD substrate or propeptide. These analyses are the first to dissect the rates of release versus carboxylation and showed that release was much slower than carboxylation. In the absence of excess VKD substrate/propeptide, fIX in the fIX-carboxylase complex was fully carboxylated by 10 min, but 95% was still complexed with carboxylase after 30 min. The presence of excess VKD substrate/propeptide, however, led to a significant increase in VKD product release, possibly through a second propeptide binding site in the carboxylase. The intracellular analyses also showed that the fIX carboxylation rate was slow in vivo and was similar in r-fIX versus r-fIX/r-carboxylase cells, despite the large differences in carboxylase levels. The results suggest that the vitamin K cofactor may be limiting for carboxylation in the cell lines.

Novel effects of diets enriched with corn oil or with an olive oil/sunflower oil mixture on vitamin K metabolism and vitamin K-dependent proteins in young men

Little is known of how the fat components of diets influence the absorption and metabolism of vitamin K and the possible consequences to the synthesis of vitamin K-dependent (VKD) proteins in different target organs. We have evaluated the effects of two diets on circulating phylloquinone (K1) and triacylglycerols (TAG). One diet was enriched with corn oil (CO) (also rich in gamma-tocopherol) and the other with an olive/sunflower (O/SO) mixture (rich in alpha-tocopherol). Effects on gamma-carboxylation were assessed from coagulation assays and sensitive assays for undercarboxylated prothrombin (ucFII) and osteocalcin (ucOC). Total plasma matrix Gla-protein (MGP) was also measured. After an initial adjustment diet, 26 healthy young men were fed, in a crossover design, the O/SO or CO diet for 2 weeks. Mean intakes of K1 during consumption of adjustment, O/SO, and CO diets were 225 microg/day, 291 microg/day, and 291 microg/day, respectively. Mean fasting levels of TAG and K1 were both significantly reduced by the CO diet, but not by the O/SO diet. Neither diet reduced FII activity but ucFII became detectable in nine subjects, eight of whom showed this abnormality with both diets. The CO diet induced a rise in ucOC (P < 0.05), which was negatively correlated to ucFII (r = -0.71, P < 0.03). The CO but not O/SO diet induced a decrease of total circulating MGP. We conclude that both oils, notably CO, affected vitamin K absorption and/or metabolism which may increase the requirements for gamma-carboxylation. The mechanism is unclear but may result from interactions of vitamin K with PUFA and/or other lipid components such as vitamin E.

The Vitamin K-dependent Carboxylase

The only known biological function of Vitamin K (Fig. 1) in animals is as a required cofactor for the production of the unusual amino acid, -carboxyglutamate (Gla). This amino acid has a profound role in human blood coagulation. Several blood proteins require the presence of nine to thirteen Gla residues for normal function; these are the socalled Vitamin K-dependent (VKD) proteins. While some of the VKD blood proteins have a pro-coagulant function (prothrombin, and factors VII, IX, and X), others primarily serve anti-coagulant roles (proteins C, S, and Z). For all of the VKD blood proteins, however, the Gla residues are located in a homologous ≈ 45 residue amino-terminal “Gla” domain (1-3). The presence of multiple Gla residues in this domain allow it to adopt a calcium-dependent conformation that promotes binding to a membrane surface (4, 5), such as found on damaged vascular endothelial cells or activated platelets. This interaction allows the localization of the VKD blood protein near the site of vascular injury, where it participates in reactions that either promote or regulate clotting (6-8). The importance of Gla in the catalytic function of these proteins is emphasized by the effect that administration of Vitamin K antagonists (e. g. coumadins, such as warfarin sodium) has on humans. Warfarin decreases the concentration of vitamin K in the tissues, which, in turn, results in the production of VKD proteins that contain a decreased number (or a complete absence) of Gla. The Gla domain of these under-carboxylated blood proteins cannot adopt its natural conformation and, as a result, the VKD blood proteins have poor affinity for phospholipid surfaces. For this reason, the reactions that involve these proteins are significantly damped, and efficient clotting no longer occurs (9-11). Gla is synthesized in mammals by post-translational modification of glutamate. The enzyme that catalyzes the conversion of glutamate to Gla is the vitamin K-dependent -glutamyl carboxylase (12). In addition to a glutamate-containing substrate, this enzyme requires carbon dioxide, reduced Vitamin K, and molecular oxygen as reactants. The products of the carboxylase-catalyzed reaction are Gla, Vitamin K 2,3 epoxide, and water (13) (Fig. 2). The enzyme is found not only in liver (where the VKD blood proteins are produced and secreted), but also in a variety of other tissues, such as skin, lung, and kidney (13, 14). The widespread tissue distribution of carboxylase in humans suggests the presence of additional Gla-containing proteins of diverse function. To date, fourteen VKD proteins have been identified in humans, seven of which are the blood proteins mentioned above. Two other proteins (bone-gla and matrix-gla protein) are involved in bone metabolism (15), while another (Gas6) is involved in cell signaling (16, 17). Screenings of human nucleotide-based databases for sequences with homology to Gla domain sequences have identified four additional putative VKD proteins of unknown function: PRGP1, PRGP2, TMG3 and TMG4 (18, 19).

Identification of Sequences within the γ-Carboxylase That Represent a Novel Contact Site with Vitamin K-dependent Proteins and That Are Required for Activity

The vitamin K-dependent (VKD) carboxylase converts clusters of Glu residues to gamma-carboxylated Glu residues (Glas) in VKD proteins, which is required for their activity. VKD precursors are targeted to the carboxylase by their carboxylase recognition site, which in most cases is a propeptide. We have identified a second tethering site for carboxylase and VKD proteins that is required for carboxylase activity, called the vitamin K-dependent protein site of interaction (VKS). Several VKD proteins specifically bound an immobilized peptide comprising amino acids 343-355 of the human carboxylase (CVYKRSRGKSGQK) but not a scrambled peptide containing the same residues in a different order. Association with the 343-355 peptide was independent of propeptide binding, because the VKD proteins lacked the propeptide and because the 343-355 peptide did not disrupt association of a propeptide factor IX-carboxylase complex. Analysis with peptides that overlapped amino acids 343-355 indicated that the 343-345 CVY residues were necessary but not sufficient for prothrombin binding. Ionic interactions were also suggested because peptide-VKD protein binding could be disrupted by changes in ionic strength or pH. Mutagenesis of Cys(343) to Ser and Tyr(345) to Phe resulted in 7-11-fold decreases in vitamin K epoxidation and peptide (EEL) substrate and carboxylase carboxylation, and kinetic analysis showed 5-6-fold increases in K(m) values for the Glu substrate. These results suggest that Cys(343) and Tyr(345) are near the catalytic center and affect the active site conformation required for correct positioning of the Glu substrate. The 343-355 VKS peptide had a higher affinity for carboxylated prothrombin (K(d) = 5 microm) than uncarboxylated prothrombin (K(d) = 60 microm), and the basic VKS region may also facilitate exiting of the Gla product from the catalytic center by ionic attraction. Tethering of VKD proteins to the carboxylase via the propeptide-binding site and the VKS region has important implications for the mechanism of VKD protein carboxylation, and a model is proposed for how the carboxylase VKS region may be required for efficient and processive VKD protein carboxylation.

Tethered processivity of the vitamin K-dependent carboxylase: factor IX is efficiently modified in a mechanism which distinguishes Gla's from Glu's and which accounts for comprehensive carboxylation in vivo.

The vitamin K-dependent (VKD) carboxylase binds VKD proteins via their propeptide and converts Glu's to gamma-carboxylated Glu's, or Gla's, in the Gla domain. Multiple carboxylation is required for activity, which could be achieved if the carboxylase is processive. In the only previous study to test for this capability, an indirect assay was used which suggested processivity; however, the efficiency was poor and raised questions regarding how full carboxylation is accomplished. To unequivocally determine if the carboxylase is processive and if it can account for comprehensive carboxylation in vivo, as well as to elucidate the enzyme mechanism, we developed a direct test for processivity. The in vitro carboxylation of a complex containing carboxylase and full-length factor IX (fIX) was challenged with an excess amount of a distinguishable fIX variant. Remarkably, carboxylation of fIX in the complex was completely unaffected by the challenge protein, and comprehensive carboxylation was achieved, showing conclusively that the carboxylase is processive and highly efficient. These studies also showed that carboxylation of individual fIX/carboxylase complexes was nonsynchronous and implicated a driving force for the reaction which requires the carboxylase to distinguish Glu's from Gla's. We found that the Gla domain is tightly associated with the carboxylase during carboxylation, blocking the access of a small peptide substrate (EEL). The studies describe the first analysis of preformed complexes, and the rate for full-length, native fIX in the complex was equivalent to that of the substrate EEL. Thus, intramolecular movement within the Gla domain to reposition new Glu's for catalysis is as rapid as diffusion-limited positioning of a small substrate, and the Gla domain is not sterically constrained by the rest of the fIX molecule during carboxylation. The rate of carboxylation of fIX in the preformed complex was 24-fold higher than for fIX modified by free carboxylase, which supports carboxylase processivity and which indicates that binding and/or release is the rate-limiting step in protein carboxylation. These data indicate a model of tethered processivity, in which the VKD proteins remain bound to the carboxylase throughout the reaction via their propeptide, while the Gla domain undergoes intramolecular movement to reposition new Glu's for catalysis to ultimately achieve comprehensive carboxylation.

Congenital Deficiency of Vitamin K-dependent Coagulation Factors – A Vitamin-K Dependency State?

Congenital Deficiency of Vitamin K-dependent Coagulation Factors A Vitamin-K Dependency State? -

The Vitamin K–Dependent Carboxylase

The carboxylase is an integral membrane glycoprotein that uses vitamin K to modify clusters of glutamyl residues (glu's) to gamma-carboxylated glutamyl residues (gla's) post-translationally in vitamin K-dependent (VKD) proteins as they pass through the endoplasmic reticulum. Carboxylation is required for VKD protein functions in hemostasis, bone metabolism, growth control and signal transduction. Carboxylation of multiple glu residues is accomplished via a processive mechanism, which occurs with at least some order and involves carboxylation of the carboxylase. The carboxylase has a high affinity binding site for VKD proteins, which in most cases is a VKD propeptide sequence; it also appears to have a low affinity site for those glu's undergoing catalysis. The propeptide activates binding of the glu's; together, the two contact points between the carboxylase and VKD protein increase the affinity of the carboxylase for vitamin K. Biochemical mapping to identify where these events occur in the carboxylase remains a challenge, despite the availability of recombinant protein. The affinity of the carboxylase for the propeptide of several VKD proteins that are coexpressed in liver varies over a 100-fold range. Treatment with anticoagulants such as warfarin that indirectly block carboxylation likely decreases the rate of VKD protein catalysis and increases the accumulation of VKD precursors, leading to a competitive state among these proteins, which results in the premature dissociation of undercarboxylated, inactive protein.

Vitamin K-dependent carboxylation of the carboxylase.

Vitamin K-dependent (VKD) proteins require modification by the VKD-gamma-glutamyl carboxylase, an enzyme that converts clusters of glus to glas in a reaction that requires vitamin K hydroquinone, for their activity. We have discovered that the carboxylase also carboxylates itself in a reaction dependent on vitamin K. When pure human recombinant carboxylase was incubated in vitro with 14CO2 and then analyzed after SDS/PAGE, a radiolabeled band corresponding to the size of the carboxylase was observed. Subsequent gla analysis of in vitro-modified carboxylase by base hydrolysis and HPLC showed that all of the radioactivity could be attributed to gla residues. Quantitation of gla, asp, and glu residues indicated 3 mol gla/mol carboxylase. Radiolabeled gla was acid-labile, confirming its identity, and was not observed if vitamin K was not included in the in vitro reaction. Carboxylase carboxylation also was detected in baculovirus-(carboxylase)-infected insect cells but not in mock-infected insect cells, which do not express endogenous VKD proteins or carboxylase. Finally, we showed that the carboxylase was carboxylated in vivo. Carboxylase was purified from recombinant carboxylase BHK cells cultured in the presence or absence of vitamin K and analyzed for gla residues. Carboxylation of the carboxylase only was observed with carboxylase isolated from BHK cells cultured in vitamin K, and 3 mol gla/mol carboxylase were detected. Analyses of carboxylase and factor IX carboxylation in vitro suggest a possible role for carboxylase carboxylation in factor IX turnover, and in vivo studies suggest a potential role in carboxylase stability. The discovery of carboxylase carboxylation has broad implications for the mechanism of VKD protein carboxylation and Warfarin-based anti-coagulant therapies that need to be considered both retrospectively and in the future.