Vitamin K-dependent carboxylation. A synthetic peptide based upon the gamma-carboxylation recognition site sequence of the prothrombin propeptide is an active substrate for the carboxylase in vitro

The vitamin K-dependent blood-clotting proteins contain a gamma-carboxylation recognition site in the propeptide, between the signal peptide and the mature protein, that directs gamma-carboxylation of specific glutamic acid residues. To develop a better substrate for the in vitro assay of the vitamin K-dependent gamma-carboxylase and to understand the substrate recognition requirements of the carboxylase, we prepared synthetic peptides based upon the structure of human proprothrombin. These peptides were employed as substrates for in vitro carboxylation using a partially purified form of the bovine liver carboxylase. A 28-residue peptide (HVFLAPQQARSLLQRVRRANTFLEEVRK), based on residues -18 to +10 in proprothrombin, includes the complete propeptide and the first 10 residues of acarboxyprothrombin. Carboxylation of this peptide is characterized by a Km of 3.6 microM. In contrast, FLEEL is carboxylated with a Km of about 2200 microM. A 10-residue peptide (ANTFLEEVRK), based on residues +1 to +10 in prothrombin, and a 20-residue peptide (ARSLLQRVRRANTFLEEVRK), based on residues -10 to +10 in proprothrombin, are also poor substrates for the carboxylase. Replacement of phenylalanine with alanine at residue 3 (equivalent to position -16 in proprothrombin) in the 28-residue peptide significantly alters the Km to 200 microM. A synthetic propeptide (HVFLAPQQARSLLQRVRRY), homologous to residues -18 to -1 in proprothrombin, inhibited carboxylation of the 28-residue peptide substrate with a Ki of 3.5 microM, but modestly stimulated the carboxylation of the 5- and 10-residue peptide substrates. These results indicate that an intact carboxylation recognition site is required for efficient in vitro carboxylation and that this site includes critical residues in region -18 to -11 of proprothrombin. The carboxylation recognition site in the propeptide binds directly to the carboxylase or to a closely associated protein.     

Identification of amino acids in the gamma-carboxylation recognition site on the propeptide of prothrombin

A gamma-carboxylation recognition site on the propeptide of the vitamin K-dependent blood coagulation proteins directs the carboxylation of glutamic acid residues by binding to the vitamin K-dependent carboxylase. To determine residues that define this site, we evaluated the effect of mutation of certain residues in the prothrombin propeptide on the extent of carboxylation. The prothrombin cDNA modified by site-specific mutagenesis was expressed in Chinese hamster ovary cells using a system that yields functional fully carboxylated prothrombin. The cell supernatants containing recombinant prothrombin were evaluated for the extent of gamma-carboxylation by immunoassay. Conformation-specific anti-prothrombin:Ca(II)-specific antibodies measure native completely carboxylated prothrombin; anti-prothrombin:total antibodies measure all forms of prothrombin, regardless of gamma-carboxyglutamic acid content. Mutation of His-18 to Gly, Val-17 to Ser, Leu-15 to Gly or Asp, or Ala-10 to Asp was associated with a partial (30-65%) inhibition of gamma-carboxylation. Mutation of Ala-14 to Ser or Ser-8 to Val did not inhibit gamma-carboxylation. From this and earlier work, residues whose mutation leads to a significant impairment of carboxylation include His-18, Val-17, Phe-16, Leu-15, and Ala-10. Residues whose mutation does not alter the carboxylation recognition site include Ala-14, Ser-8, Arg-4, and Arg-1. To determine the size of the recognition site, the in vitro carboxylation of propeptide-containing synthetic peptides was compared. A 28-residue peptide, based upon residues -18 to +10 of prothrombin, and a 54-residue peptide, based upon residues -18 to +36 of prothrombin, were carboxylated by partially purified bovine carboxylase with similar Km values of 2-5 microM. These results indicate that the gamma-carboxyglutamic acid-rich region of prothrombin makes a minimal contribution to carboxylase binding. A molecular surface of about five amino acids located within the propeptide appears to define the carboxylation recognition site on the precursor forms of the vitamin K-dependent proteins.     

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.     

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.     

Vitamin K-dependent carboxylation. In vitro modification of synthetic peptides containing the gamma-carboxylation recognition site

Synthetic peptides including the gamma-carboxylation recognition site and acidic amino acids were compared as substrates for vitamin K-dependent gamma-carboxylation by bovine liver carboxylase. The 28-residue proPT28 (proprothrombin -18 to +10) and proFIX28 (pro-Factor IX -18 to +10) were carboxylated with a Km of 3 microM. The Vmax of proPT28 was 2-3 times greater than that of proFIX28. An analog of proFIX28 that contained the prothrombin propeptide had a Vmax 2-3-fold greater than an analog of proPT28 that contained the Factor IX propeptide. proFIX28/RS-1, based upon Factor IX Cambridge, proFIX28/RQ-4, based upon Factor IX Oxford 3, and proFIX28 had equivalent Km and Vmax values. Analogs of proPT28 containing Ala6-Glu7 or Glu6-Ala7 were carboxylated at equivalent rates. A peptide containing Asp6-Asp7 was carboxylated at a rate of about 1% of that of Glu carboxylation. Carboxylation of peptides containing Asp6-Glu7 and Glu6-Asp7 yielded results identical with peptides containing Ala6-Glu7 and Glu6-Ala7. Carboxymethylcysteine was not carboxylated when substituted for Glu6 in a peptide containing Asp7. These results indicate that the prothrombin propeptide is more efficient in the carboxylation process than is the Factor IX propeptide, but that both propeptides direct carboxylation; the gamma-carboxylation recognition site does not include residues -4 and -1; aspartic acid and carboxymethylcysteine are poor substrates for the carboxylase, but aspartic acid does not inhibit the carboxylation of adjacent glutamic acids.     

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.     

Propeptide recognition by the vitamin K-dependent carboxylase in early processing of prothrombin and factor X.

Precursors of vitamin K-dependent proteins are synthesized with a propeptide that is believed to target these proteins for gamma-carboxylation by the vitamin K-dependent carboxylase. In this study synthetic propeptides were used to investigate gamma-carboxylation of the prothrombin and factor X precursors in rat liver microsomes. The extent of prothrombin processing by the carboxylase was also investigated. Antisera raised against the human prothrombin and factor X propeptides only recognized precursors with the respective propeptide regions. The data demonstrate structural differences in the propeptide region of the prothrombin and the factor X carboxylase substrates which raises questions about the hypothesis of a common propeptide binding site on the carboxylase for all precursors of vitamin K-dependent proteins. The hypothesis of separate binding sites is supported by data which demonstrate differences in binding of the prothrombin and factor X precursors to membrane fragments from rough and smooth microsomes. gamma-Carboxylation of the prothrombin precursors in vitro was investigated with conformational specific antibodies raised against a portion of the Gla (gamma-carboxyglutamic acid) region extending from residue 15 to 24. The synthetic peptide used as antigen contains three of the ten potential Gla sites in prothrombin. It is shown that these antibodies do not recognize mature prothrombin but recognize the decarboxylated protein. It is also demonstrated that the epitope is Ca2(+)-dependent. The antibodies were used to assess gamma-carboxylation of the prothrombin precursor in membrane fragments from microsomal membranes. The results suggest that microsomal gamma-carboxylation does not involve Glu residues 16, 19 and 20 of the Gla region.     

Hydrophobic amino acids define the carboxylation recognition site in the precursor of the gamma-carboxyglutamic-acid-containing conotoxin epsilon-TxIX from the marine cone snail Conus textile.

To identify the amino acid sequence of the precursor of the Gla-containing peptide, epsilon-TxIX, from the venom of the marine snail Conus textile, the cDNA encoding this peptide was cloned from a C. textile venom duct library. The cDNA of the precursor form of epsilon-TxIX encodes a 67 amino acid precursor peptide, including an N-terminal prepro-region, the mature peptide, and four residues posttranslationally cleaved from the C-terminus. To determine the role of the propeptide in gamma-carboxylation, peptides were designed and synthesized based on the propeptide sequence of the Gla-containing conotoxin epsilon-TxIX and used in assays with the vitamin K-dependent gamma-glutamyl carboxylase from C. textile venom ducts. The mature acarboxy peptide epsilon-TxIX was a high K(M) substrate for the gamma-carboxylase. Synthetic peptides based on the precursor epsilon-TxIX were low K(M) substrates (5 microM) if the peptides included at least 12 residues of propeptide sequence, from -12 to -1. Leucine-19, leucine-16, asparagine-13, leucine-12, leucine-8 and leucine-4 contribute to the interaction of the pro-conotoxin with carboxylase since their replacement by aspartic acid increased the K(M) of the substrate peptide. Although the Conus propeptide and the propeptides of the mammalian vitamin K-dependent proteins show no obvious sequence homology, synthetic peptides based upon the structure of pro-epsilon-TxIX were intermediate K(M) substrates for the bovine carboxylase. The propeptide of epsilon-TxIX contains significant alpha-helix, as estimated by measurement of the circular dichroism spectra, but the region of the propeptide that plays the dominant role in directing carboxylation does not contain evidence of helical structure. These results indicate that the gamma-carboxylation recognition site is defined by hydrophobic residues in the propeptide of this conotoxin precursor.     

Effect of propeptide amino acid substitution in γ‐carboxylation,activity and expression of recombinant human coagulation factor IX

The production of recombinant vitamin K dependent (VKD) proteins for therapeutic purposes is an important challenge in the pharmaceutical industry. These proteins are primarily synthesized as precursor molecules and contain pre–propeptide sequences. The propeptide is connected to γ‐carboxylase enzyme through the γ‐carboxylase recognition site for the direct γ‐carboxylation of VKD proteins that has a significant impact on their biological activity. Propeptides have different attitudes toward γ‐carboxylase and certain amino acids in propeptide sequences are responsible for the differences in γ‐carboxylase affinity. By aiming to replace amino acids in hFIX propeptide domain based on the prothrombin propeptide, pMT‐hFIX‐M14 expression cassette, containing cDNA of hFIX with substituted ?14 residues (Asp to Ala) was made. After transfection of Drosophila S2 cells, expression of the active hFIX was analyzed by performing ELISA and coagulation test. A 1.4‐fold increase in the mutant recombinant hFIX expression level was observed in comparison with that of a native recombinant hFIX. The enhanced hFIX activity and specific activity of the hFIXD‐14A (2.2 and 1.6 times, respectively) were further confirmed by comparing coagulation activity levels of substituted and native hFIX. Enrichment for functional, fully γ‐carboxylated hFIX species via barium citrate adsorption demonstrated 2‐fold enhanced recovery in the S2‐expressing hFIXD‐14A relative to that expressed native hFIX. These results show that changing ?14 residues leads to a decrease in the binding affinity to substrate, increase in γ‐carboxylation and activity of recombinant hFIX. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:515–520, 2018     

Vitamin K-dependent carboxylase: effect of detergent concentrations, vitamin K status, and added protein precursors on activity

Activity of the rat liver microsomal vitamin K-dependent carboxylase has been studied at various concentrations of detergent. The activity which could be solubilized by 0.25% Triton X-100 was low but could be greatly increased if vitamin K-deficient rats were given vitamin K a few minutes before they were killed. At higher concentrations of Triton, more activity was solubilized and this effect was not seen. In vitro carboxylation of endogenous microsomal proteins was decreased by 80-90% if vitamin K was administered 1 min before rats were killed, but the amount of assayable prothrombin precursor was decreased by only 20%. Decarboxylated vitamin K-dependent rat plasma proteins were not substrates for the carboxylase and did not influence peptide carboxylase activity significantly. Purified microsomal prothrombin precursors did, however, stimulate carboxylation of peptide substrate and were used as a substrate for the carboxylase in a preparation from precursor depleted vitamin K-deficient rats.     

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.     

The propeptides of the vitamin K-dependent proteins possess different affinities for the vitamin K-dependent carboxylase.

The vitamin K-dependent gamma-glutamyl carboxylase catalyzes the modification of specific glutamates in a number of proteins required for blood coagulation and associated with bone and calcium homeostasis. All known vitamin K-dependent proteins possess a conserved eighteen-amino acid propeptide sequence that is the primary binding site for the carboxylase. We compared the relative affinities of synthetic propeptides of nine human vitamin K-dependent proteins by determining the inhibition constants (Ki) toward a factor IX propeptide/gamma-carboxyglutamic acid domain substrate. The Ki values for six of the propeptides (factor X, matrix Gla protein, factor VII, factor IX, PRGP1, and protein S) were between 2-35 nM, with the factor X propeptide having the tightest affinity. In contrast, the inhibition constants for the propeptides of prothrombin and protein C are approximately 100-fold weaker than the factor X propeptide. The propeptide of bone Gla protein demonstrates severely impaired carboxylase binding with an inhibition constant of at least 200,000-fold weaker than the factor X propeptide. This study demonstrates that the affinities of the propeptides of the vitamin K-dependent proteins vary over a considerable range; this may have important physiological consequences in the levels of vitamin K-dependent proteins and the biochemical mechanism by which these substrates are modified by the carboxylase.     

Vitamin K-dependent carboxylase: requirements for carboxylation of soluble peptide and substrate specificity.

Rat liver microsomes contain a triton X-100 solubilizable vitamin K-dependent carboxylase activity that converts specific glutamyl residues of precursor proteins to γ-carboxyglutamyl residues. This activity has been studied utilizing synthetic peptides as substrates for the enzyme. When compared to the carboxylation of the endogenous microsomal precursors, the peptide carboxylase activity is more sensitive to the action of various inhibitors, and requires a higher concentration of vitamin K for maximal activity. The apparent K_m for the peptide Phe-Leu-Glu-Glu-Leu was found to be 4 mM. Substrate specificity depends on residues adjacent to the carboxylated Glu residues and macromolecular recognition sites.     

Effect of Vitamin K-dependent Protein Precursor Propeptide,Vitamin K Hydroquinone,and Glutamate Substrate Binding on the Structure and Function of γ-Glutamyl Carboxylase

The γ-glutamyl carboxylase utilizes four substrates to catalyze carboxylation of certain glutamic acid residues in vitamin K-dependent proteins. How the enzyme brings the substrates together to promote catalysis is an important question in understanding the structure and function of this enzyme. The propeptide is the primary binding site of the vitamin K-dependent proteins to carboxylase. It is also an effector of carboxylase activity. We tested the hypothesis that binding of substrates causes changes to the carboxylase and in turn to the substrate-enzyme interactions. In addition we investigated how the sequences of the propeptides affected the substrate-enzyme interaction. To study these questions we employed fluorescently labeled propeptides to measure affinity for the carboxylase. We also measured the ability of several propeptides to increase carboxylase catalytic activity. Finally we determined the effect of substrates: vitamin K hydroquinone, the pentapeptide FLEEL, and NaHCO₃, on the stability of the propeptide-carboxylase complexes. We found a wide variation in the propeptide affinities for carboxylase. In contrast, the propeptides tested had similar effects on carboxylase catalytic activity. FLEEL and vitamin K hydroquinone both stabilized the propeptide-carboxylase complex. The two together had a greater effect than either alone. We conclude that the effect of propeptide and substrates on carboxylase controls the order of substrate binding in such a way as to ensure efficient, specific carboxylation.     

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, Br?nsted 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.     

Vitamin K-dependent carboxylase: influence of the "propeptide" region on enzyme activity

The liver microsomal vitamin K-dependent carboxylase catalyzes the post-translational conversion of specific glutamyl to gamma-carboxyglutamyl (Gla) residues in precursor forms of a limited number of proteins. These proteins contain an amino-terminal extension (propeptide) that is presumed to serve as an enzyme recognition site to assure their normal processing. The free, noncovalently bound propeptide has also been shown to stimulate the in vitro activity of this enzyme. This peptide has now been shown to lower the app Km of a low-molecular-weight Glu site substrate while having no influence on the app Km of the other substrates, vitamin KH2, O2, and CO2/HCO3-. Propeptide addition was shown to have no influence on the ratio of the two products of the enzyme, Gla and vitamin K-2,3-epoxide. Stimulation of carboxylase activity by the propeptide from human factor X was observed in a number of rat tissues and in the liver of a number of different species. Stability of the enzyme in crude microsomal preparations was greatly enhanced by the presence of propeptide. These observations are consistent with the hypothesis that this region of the protein substrates for the carboxylase not only serves an enzyme recognition or docking function but also modulates the activity of the enzyme by altering the affinity for one of its substrates.     

Vitamin K-dependent carboxylation of glutamic acid residues to γ-carboxyglutamic acid in lung microsomes

Vitamin K-dependent carboxylation of glutamic acid residues to γ-carboxyglutamic acid was demonstrated in proteins of lung microsomes. The carboxylation was 12% of that in liver microsomes per milligram of mierosomal protein. Carboxylation was very low with microsomes of untreated rats but increased with time up to 42 h after warfarin administration. Carboxylation was highest with microsomes from rats fed a vitamin K-deficient diet. This suggests that a protein(s) accumulates which can be carboxylated in vitro/J. Lung microsomes also catalyzed the vitamin K-dependent carboxylation of the peptide Phe-Leu-Glu-Glu-Leu. The peptide carboxylase activity was 9% of that obtained with liver microsomes. Vitamin K-dependent protein carboxylation required NADH or dithioerythritol, suggesting that vitamin K had to be reduced to the hydroquinone. Accordingly, vitamin K₁ hydroquinone had carboxylating activity without added reducing agents. Menaquinone-3 was considerably more active than phylloquinone. The temperature optimum for carboxylation was around 27 °C.     

A novel fluorescence assay to study propeptide interaction with gamma-glutamyl carboxylase

The vitamin K-dependent gamma-glutamyl carboxylase catalyzes the posttranslational modification of select glutamate residues of its vitamin K-dependent substrates to gamma-carboxyglutamate. In this report, we describe a new fluorescence assay that is sensitive and specific for the propeptide binding site of active carboxylase. We employed the assay to make three important observations: (1) A tight binding fluorescein-labeled consensus propeptide can be used to quantify the active fraction of the enzyme. (2) The off-rate for a fluorescein-labeled factor IX propeptide was 3000-fold slower than the rate of carboxylation, a difference that may explain how carboxylase can carry out multiple carboxylations of a substrate during the same binding event. (3) We show evidence that substrate binding to the active site modifies the propeptide binding site of carboxylase. The significant (9-fold) differences in off-rates for the propeptide in the presence and absence of its co-substrates may represent a release mechanism for macromolecular substrates from the enzyme. Additionally, sedimentation velocity and equilibrium experiments indicate a monomeric association of enzyme with propeptide. Furthermore, the carboxylase preparation is monodisperse in the buffer used for our studies.     

The putative vitamin K-dependent gamma-glutamyl carboxylase internal propeptide appears to be the propeptide binding site

The vitamin K-dependent gamma-glutamyl carboxylase binds an 18-amino acid sequence usually attached as a propeptide to its substrates. Price and Williamson (Protein Sci. (1993) 2, 1997-1998) noticed that residues 495-513 of the carboxylase shares similarity with the propeptide. They suggested that this internal propeptide could bind intramolecularly to the propeptide binding site of carboxylase, thereby preventing carboxylation of substrates lacking a propeptide recognition sequence. To test Price's hypothesis, we created nine mutant enzyme species that have single or double mutations within this putative internal propeptide. The apparent K(d) values of these mutant enzymes for human factor IX propeptide varied from 0.5- to 287-fold when compared with that of wild type enzyme. These results are consistent with the internal propeptide hypothesis but could also be explained by these residues participating in propeptide binding site per se. To distinguish between the two alternative hypotheses, we measured the dissociation rates of propeptides from each of the mutant enzymes. Changes in an internal propeptide should not affect the dissociation rates, but changes to a propeptide binding site may affect the dissociation rate. We found that dissociation rates varied in a manner consistent with the apparent K(d) values measured above. Furthermore, kinetic studies using propeptide-containing substrates demonstrated a correlation between the affinity for propeptide and V(max). Taken together, our results indicated that these mutations affected the propeptide binding site rather than a competitive inhibitory internal propeptide sequence. These results agree with our previous observations, indicating that residues in this region are involved in propeptide binding.     

Recognition site directing vitamin K-dependent gamma-carboxylation resides on the propeptide of factor IX

Posttranslational processing of vitamin K-dependent proteins includes gamma-carboxylation of specific glutamic acid residues to form gamma-carboxyglutamic acids. To determine whether carboxylation is directed by the propeptide sequence, homologous among the precursors of these proteins, alterations were made in the Factor IX propeptide cDNA. The extent of gamma-carboxylation of recombinant Factor IX was assessed using conformation-specific antibodies directed against the gamma-carboxyglutamic acid-dependent, metal-stabilized structure. Deletion of the propeptide (residues -18 to -1) abolished carboxylation, but not secretion, of Factor IX. Substitution of alanine for phenylalanine -16 or glutamic acid for alanine -10 also impaired carboxylation. These results indicate that the Factor IX propeptide participates in defining a recognition site that designates an adjacent glutamic acid-rich domain for gamma-carboxylation. The association of the propeptide with the gamma-carboxylation recognition site provides the first demonstration of a specific function served by a propeptide in posttranslational protein processing.