Vitamin K-dependent carboxylase: Synthesis of an inhibitor of the glutamyl binding site

Liver microsomes contain a vitamin K and O₂-dependent carboxylase that converts peptide-bound glutamyl residues to γ-carboxyglutamyl residues. The peptide Boc-O-phospho—Ser-O-phospho—Ser—Leu-OMe has now been synthesized. This peptide inhibits the carboxylation of endogenous protein precursors by a detergent-solubilized preparation of the carboxylase and is an apparent competitive inhibitor of the carboxylation of Phe—Leu—Glu—Glu—Leu.     

Identification of sequences within the gamma-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.     

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.     

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.     

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.     

Vitamin K-dependent carboxylase: effect of endogenous microsomal protein precursors on the rate of exogenous substrate carboxylation

Rat liver microsomes contain a Triton X-100 solubilizable vitamin K-dependent carboxylase activity that converts specific glutamyl residues of a microsomal prothrombin precursor to gamma-carboxyglutamyl residues. This activity has been studied in partially (0.25% Triton X-100) and completely (1.0% Triton X-100) solubilized rat liver microsomal preparations. The rate of vitamin K-dependent carboxylation of endogenous microsomal protein precursors was very rapid in the completely solubilized liver microsomal preparation, and carboxylation of an exogenous peptide substrate (Phe-Leu-Glu-Glu-Leu) proceeded at the same time. In the partially solubilized liver microsomal preparation, the rate of protein carboxylation was greatly reduced, and a lag in carboxylation of the exogenous substrate was observed. When microsomal preparations which were depleted of endogenous precursors were used, this lag was eliminated. These data suggest that both substrates utilize the same microsomal pool of carboxylase and that the fraction of the carboxylase bound to the endogenous precursors is not immediately available to exogenous substrates.     

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.     

Vitamin K-dependent carboxylase. Requirements of the rat liver microsomal enzyme system.

Vitamin K is required in an enzymatic reaction which carboxylates glutamyl residues in a microsomal protein precursor of plasma prothrombin to form gamma-carboxyglutamic acid residues. The partial requirements of this microsomal, vitamin K-dependent carboxylase system have been determined. A requirement of the system for cytosolic factors appears to be due primarily to the presence of reduced pyridine nucleotides or a reduced pyridine nucleotide-generating system in the cytosol. The hydroquinone of vitamin K has been demonstrated to be the enzymatically active form of the vitamin. When vitamin K1 hydroquinone is added to the carboxylase system, no NAD(P)H is needed for maximum activity. The carboxylase activity is half-maximally stimulated by 0.25 mug of vitamin K1/ml in the presence of cytosolic components but requires at least 10 times as much vitamin when microsomes are incubated in a cytosol-free buffer. Menadione is inactive as a vitamin source in this system, and the carboxylase activity is inhibited by the 2-chloro analog of vitamin K1 and by Warfarin. The ATP analog, AMP-P(NH)P, inhibited the carboxylase activity, but a dependence on exogenous ATP or an ATP-generating system could not be demonstrated. Carboxylase activity was found to be dependent on an O2-containing gas phase, and upon the HCO3- concentration.     

Characterization of vitamin K-dependent carboxylase from the livers from the adult ox and dicoumarol-treated calf.

The properties of the microsomal vitamin K-dependent carboxylase from the livers of the adult ox and dicoumarol-treated calf were investigated. The enzymes from both sources utilized glutamic residues of synthetic peptides as substrates and could be solubilized with Triton X-100 similarly to the enzyme from vitamin K-deficient rat liver. Under the optimal assay conditions, the microsomes from calf liver had peptide carboxylase activity comparable with that of the rat liver microsomes and 6.5-fold that of adult ox liver microsomes. The apparent Km for reduced vitamin K and the ionic strength optima of the calf and adult ox enzyme clearly differ from those of the rat enzyme. Pyridoxal phosphate activated the adult ox carboxylase only slightly, whereas the calf enzyme was activated by pyridoxal phosphate as effectively as was the enzyme from the vitamin K-deficient rat. Mn2+ activated the adult ox enzyme 9-fold and calf enzyme 22-fold under optimal conditions (no KCl). Three other divalent metal cations (Ca2+, Ba2+, and Mg2+) activated the adult ox and calf enzymes to about half the extent caused by Mn2+, KCl inhibited this activation. The vitamin K-dependent carboxylase from the dicoumarol-treated calf is apparently more tightly bound to the microsomal membrane than is the adult ox enzyme. In many other respects (pH optimum), temperature optimum, Km values for peptide substrate, substrate specificity, inhibitor effects), the properties of the adult ox and calf enzymes resemble closely those of the rat enzyme.     

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.     

Chemical modification of cysteine residues is a misleading indicator of their status as active site residues in the vitamin K-dependent gamma-glutamyl carboxylation reaction

The enzymatic activity of the vitamin K-dependent proteins requires the post-translational conversion of specific glutamic acids to gamma-carboxy-glutamic acid by the integral membrane enzyme, gamma-glutamyl carboxylase. Whether or not cysteine residues are important for carboxylase activity has been the subject of a number of studies. In the present study we used carboxylase with point mutations at cysteines, chemical modification, and mass spectrometry to examine this question. Mutation of any of the free cysteine residues to alanine or serine had little effect on carboxylase activity, although C343A mutant carboxylase had only 38% activity compared with that of wild type. In contrast, treatment with either thiol-reactive reagent 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, disodium salt, or sodium tetrathionate, caused complete loss of activity. We identified the residues modified, using matrix-assisted laser desorption/ionization time of flight mass spectrometry, as Cys(323) and Cys(343). According to our results, these residues are on the cytoplasmic side of the microsomal membrane, whereas catalytic residues are expected to be on the lumenal side of the membrane. Carboxylase was partially protected from chemical modification by factor IXs propeptide. Although all mutant carboxylases bound propeptide with normal affinity, chemical modification caused a >100-fold decrease in carboxylase affinity for the consensus propeptide. We conclude that cysteine residues are not directly involved in carboxylase catalysis, but chemical modification of Cys(323) and Cys(343) may disrupt the three-dimensional structure, resulting in inactivation.     

Vitamin K-dependent carboxylase. Control of enzyme activity by the "propeptide" region of factor X

A liver microsomal enzyme catalyzes the vitamin K-dependent posttranslational carboxylation of specific glutamyl residues of a limited number of plasma proteins to gamma-carboxyglutamyl residues. The intracellular precursor forms of these proteins are known to contain a homologous basic amino acid-rich propeptide region between the signal peptide region and the amino terminus of the mature protein. This region of the precursor protein has been implicated as a possible recognition site for the carboxylase enzyme. A 20-residue peptide containing the octadecapropeptide of human clotting factor X has now been shown to strongly stimulate the activity of the enzyme toward a noncovalently linked substrate. This stimulatory effect is seen at less than micromolar concentrations and is accompanied by a decrease in the Km of the glutamic acid substrate. These observations raise the possibility that the catalytic activity of other enzymes involved in protein processing may be regulated by a portion of their normal substrates.     

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.     

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.     

Expression and characterization of recombinant vitamin K-dependent gamma-glutamyl carboxylase from an invertebrate, Conus textile.

The marine snail Conus is the sole invertebrate wherein both the vitamin K-dependent carboxylase and its product, gamma-carboxyglutamic acid, have been identified. To examine its biosynthesis of gamma-carboxyglutamic acid, we studied the carboxylase from Conus venom ducts. The carboxylase cDNA from Conus textile has an ORF that encodes a 811-amino-acid protein which exhibits sequence similarity to the vertebrate carboxylases, with 41% identity and approximately 60% sequence similarity to the bovine carboxylase. Expression of this cDNA in COS cells or insect cells yielded vitamin K-dependent carboxylase activity and vitamin K-dependent epoxidase activity. The recombinant carboxylase has a molecular mass of approximately 130 kDa. The recombinant Conus carboxylase carboxylated Phe-Leu-Glu-Glu-Leu and the 28-residue peptides based on residues -18 to +10 of human proprothrombin and proFactor IX with Km values of 420 micro m, 1.7 micro m and 6 micro m, respectively; the Km for vitamin K is 52 micro m. The Km values for peptides based on the sequence of the conotoxin epsilon-TxIX and two precursor analogs containing 12 or 29 amino acids of the propeptide region are 565 micro m, 75 micro m and 74 micro m, respectively. The recombinant Conus carboxylase, in the absence of endogenous substrates, is stimulated up to fivefold by vertebrate propeptides but not by Conus propeptides. These results suggest two propeptide-binding sites in the carboxylase, one that binds the Conus and vertebrate propeptides and is required for substrate binding, and the other that binds only the vertebrate propeptide and is required for enzyme stimulation. The marked functional and structural similarities between the Conus carboxylase and vertebrate vitamin K-dependent gamma-carboxylases argue for conservation of a vitamin K-dependent carboxylase across animal species and the importance of gamma-carboxyglutamic acid synthesis in diverse biological systems.     

Transmembrane domain interactions and residue proline 378 are essential for proper structure, especially disulfide bond formation, in the human vitamin K-dependent gamma-glutamyl carboxylase

We used recombinant techniques to create a two-chain form (residues 1-345 and residues 346-758) of the vitamin K-dependent gamma-glutamyl carboxylase, a glycoprotein located in the endoplasmic reticulum containing five transmembrane domains. The two-chain carboxylase had carboxylase and epoxidase activities similar to those of one-chain carboxylase. In addition, it had normal affinity for the propeptide of factor IX. We employed this molecule to investigate formation of the one disulfide bond in carboxylase, the transmembrane structure of carboxylase, and the potential interactions among the carboxylase's transmembrane domains. Our results indicate that the two peptides of the two-chain carboxylase are joined by a disulfide bond. Proline 378 is important for the structure necessary for disulfide formation. Results with the P378L carboxylase indicate that noncovalent bonds maintain the two-chain structure even when the disulfide bond is disrupted. As we had previously proposed, the fifth transmembrane domain of carboxylase is the last and only transmembrane domain in the C-terminal peptide of the two-chain carboxylase. We show that the noncovalent association between the two chains of carboxylase involves an interaction between the fifth transmembrane domain and the second transmembrane domain. Results of a homology model of transmembrane domains 2 and 5 suggest that not only do these two domains associate but that transmembrane domain 2 may interact with another transmembrane domain. This latter interaction may be mediated at least in part by a motif of glycine residues in the second transmembrane domain.     

Vitamin K dependent in vitro production of prothrombin

During prothrombin biosynthesis, glutamyl residues in prothrombin precursor proteins are carboxylated to gamma-carboxyglutamyl residues by a vitamin K dependent carboxylase. Calcium-dependent and calcium-independent rat prothrombin antibody subpopulations have been produced and utilized to study the liver microsomal precursors of prothrombin that accumulate when vitamin K action is blocked. A substantial portion of the precursor pool accumulating in the vitamin K deficient or warfarin-treated rat will react with a Ca2+-dependent antibody at high calcium concentration and appears to be partially carboxylated. During in vitro incubation in the presence of vitamin K, the fraction of the precursor pool which is tightly bound to the microsomal membrane appears to be the preferred substrate for the vitamin K dependent carboxylation. A small amount of completely carboxylated rather than a large amount of partially carboxylated products are produced during these incubations. Treatment with a Sepharose-bound prothrombin antibody demonstrated that about 20-25% of the total carboxylated microsomal protein precursor pool consists of prothrombin precursors. This treatment removes an equal amount of total carboxylase activity, and the enzyme is active in this carboxylase precursor-antibody complex.     

Glutamyl substrate-induced exposure of a free cysteine residue in the vitamin K-dependent gamma-glutamyl carboxylase is critical for vitamin K epoxidation.

The vitamin K-dependent carboxylase catalyzes the posttranslational modification of glutamic acid to gamma-carboxyglutamic acid in the vitamin K-dependent proteins of blood and bone. The vitamin K-dependent carboxylase also catalyzes the epoxidation of vitamin K hydroquinone, an obligatory step in gamma-carboxylation. Using recombinant vitamin K-dependent carboxylase, purified in the absence of propeptide and glutamic acid-containing substrate using a FLAG epitope tag, the role of free cysteine residues in these reactions was examined. Incubation of the vitamin K-dependent carboxylase with the sulfhydryl-reactive reagent N-ethylmaleimide inhibited both the carboxylase and epoxidase activities of the enzyme. This inhibition was proportional to the incorporation of radiolabeled N-ethylmaleimide. Stoichiometric analyses using [(3)H]-N-ethylmaleimide indicated that the vitamin K-dependent carboxylase contains two or three free cysteine residues. Incubation with propeptide, glutamic acid-containing substrate, and vitamin K hydroquinone, alone or in combination, indicated that the binding of a glutamic acid-containing substrate to the carboxylase makes accessible a free cysteine residue that is important for interaction with vitamin K hydroquinone. This is consistent with our previous observation that binding of a glutamic acid-containing substrate activates vitamin K epoxidation and supports the hypothesis that binding of the carboxylatable substrate to the enzyme results in a conformational change which renders the enzyme catalytically competent.     

Vitamin K-dependent carboxylase: minimized escape of CO2 from solution may prolong linearity of the reaction rate

Escape of 14CO2 from the reaction mixture into the gas phase may seriously affect the accuracy of in vitro measurement of vitamin K-dependent carboxylase activity (and probably that of other carboxylases as well). In this paper we describe the effect of (a) the volume of the test tubes in which the reaction is performed, (b) the addition of an excess of NaH12CO3 in parallel with standard amounts of NaH14CO3, and (c) the incubation temperature. In this way optimal conditions are defined and used for the carboxylation of various peptide and protein substrates. It is shown that both a prosequence and an internal recognition site contribute to the effective recognition of a substrate by carboxylase. The maximal efficiency of carboxylation was 1-2% with substrates lacking both signals and 20-50% if only one was present. This indicates the need for developing peptide substrates containing both recognition signals for vitamin K-dependent carboxylase.     

Protection of tryptic-sensitive sites in the large subunit of ribulosebisphosphate carboxylase/oxygenase by catalysis

Limited tryptic proteolysis of spinach (Spinacia oleracea) ribulose bisphosphate carboxylase/oxygenase (ribulose-P₂ carboxylase) resulted in the ordered release of two adjacent N-terminal peptides from the large subunit, and an irreversible, partial inactivation of catalysis. The two peptides were identified as the N-terminal tryptic peptide (acetylated Pro-3 to Lys-8) and the penultimate tryptic peptide (Ala-9 to Lys-14). Kinetic comparison of hydrolysis at Lys-8 and Lys-14, enzyme inactivation, and changes in the molecular weight of the large subunit, indicated that proteolysis at Lys-14 correlated with inactivation, while proteolysis at Lys-8 occurred much more rapidly. Thus, enzyme inactivation is primarily the result of proteolysis at Lys-14. Proteolysis of ribulose-P₂ carboxylase under catalytic conditions (in the presence of CO₂, Mg²⁺, and ribulose-P₂) also resulted in ordered release of these tryptic peptides; however, the rate of proteolysis at lysyl residues 8 and 14 was reduced to approximately one-third of the rate of proteolysis of these lysyl residues under noncatalytic conditions (in the presence of CO₂ and Mg²⁺ only). The protection of these lysyl residues from proteolysis under catalytic conditions could reflect conformational changes in the N-terminal domain of the large subunit which occur during the catalytic cycle.