GGCX mutants that impair hemostasis reveal the importance of processivity and full carboxylation to VKD protein function.

γ-Glutamyl carboxylase (GGCX) generates multiple carboxylated Glus (Glas) in vitamin K-dependent (VKD) proteins that are required for their functions. GGCX is processive, remaining bound to VKD proteins throughout multiple Glu carboxylations, and this study reveals the essentiality of processivity to VKD protein function. GGCX mutants (V255M and S300F) whose combined heterozygosity in a patient causes defective clotting and calcification were studied using a novel assay that mimics in vivo carboxylation. Complexes between variant carboxylases and VKD proteins important to hemostasis (factor IX [FIX]) or calcification (matrix Gla protein [MGP]) were reacted in the presence of a challenge VKD protein that could potentially interfere with carboxylation of the VKD protein in the complex. The VKD protein in the complex with wild-type carboxylase was carboxylated before challenge protein carboxylation occurred and became fully carboxylated. In contrast, the V255M mutant carboxylated both forms at the same time and did not completely carboxylate FIX in the complex. S300F carboxylation was poor with both FIX and MGP. Additional studies analyzed FIX- and MGP-derived peptides containing the Gla domain linked to sequences that mediate carboxylase binding. The total amount of carboxylated peptide generated by the V255M mutant was higher than that of wild-type GGCX; however, the individual peptides were partially carboxylated. Analysis of the V255M mutant in FIX HEK293 cells lacking endogenous GGCX revealed poor FIX clotting activity. This study shows that disrupted processivity causes disease and explains the defect in the patient. Kinetic analyses also suggest that disrupted processivity may occur in wild-type carboxylase under some conditions (eg, warfarin therapy or vitamin K deficiency).

Exon 2 skipping eliminates γ-glutamyl carboxylase activity, indicating a partial splicing defect in a patient with vitamin K clotting factor deficiency.

Essentials A carboxylase mutation that impairs splicing to delete exon 2 sequences was previously reported. We found that the mutant was inactive for vitamin K-dependent (VKD) protein carboxylation. An incomplete splicing defect likely accounts for VKD clotting activity observed in the patient. The results indicate the importance of proper carboxylase embedment in the membrane for function. BACKGROUND: Mutations in the γ-glutamyl carboxylase (GGCX), which is required for vitamin K-dependent (VKD) protein activation, can result in vitamin K clotting factor deficiency (VKCFD1). A recent report described a VKCFD1 patient with a homozygous carboxylase mutation that altered splicing and deleted exon 2 (Δ2GGCX). Only Δ2GGCX RNA was observed in the patient. OBJECTIVES: Loss of exon 2 results in the deletion of carboxylase sequences thought to be important for membrane topology and consequent function. Carboxylase activity is required for life, and we therefore tested whether the Δ2GGCX mutant is active. METHODS: HEK 293 cells were edited by the use of CRISPR-Cas9 to eliminate endogenous carboxylase. Recombinant wild-type GGCX and recombinant Δ2GGCX were then expressed and tested for carboxylation of the VKD protein factor IX. A second approach was used to monitor carboxylation biochemically, using recombinant carboxylases expressed in insect cells that lack endogenous carboxylase. RESULTS AND CONCLUSIONS: Δ2GGCX activity was undetectable in both assays, which is strikingly different from the low levels of carboxylase activity observed with other VKCFD1 mutants. The similarity in clotting function between patients with Δ2GGCX and these mutations must therefore arise from a novel mechanism. Low levels of properly spliced carboxylase RNA that produce full-length protein would not have been observed in the previous study. The results suggest that the splicing defect is incomplete. Δ2GGCX RNA has been detected in normal human liver, and has been designated carboxylase isoform 2; however, Δ2GGCX protein was not observed in normal human liver. The lack of activity and protein expression suggest that isoform 2 is not physiologically relevant to normal VKD protein carboxylation.

Warfarin alters vitamin K metabolism: a surprising mechanism of VKORC1 uncoupling necessitates an additional reductase.

The anticoagulant warfarin inhibits the vitamin K oxidoreductase (VKORC1), which generates vitamin K hydroquinone (KH2) required for the carboxylation and consequent activation of vitamin K-dependent (VKD) proteins. VKORC1 produces KH2 in 2 reactions: reduction of vitamin K epoxide (KO) to quinone (K), and then KH2 Our dissection of full reduction vs the individual reactions revealed a surprising mechanism of warfarin inhibition. Warfarin inhibition of KO to K reduction and carboxylation that requires full reduction were compared in wild-type VKORC1 or mutants (Y139H, Y139F) that cause warfarin resistance. Carboxylation was much more strongly inhibited (∼400-fold) than KO reduction (two- to threefold). The K to KH2 reaction was analyzed using low K concentrations that result from inhibition of KO to K. Carboxylation that required only K to KH2 reduction was inhibited much less than observed with the KO substrate that requires full VKORC1 reduction (eg, 2.5-fold vs 70-fold, respectively, in cells expressing wild-type VKORC1 and factor IX). The results indicate that warfarin uncouples the 2 reactions that fully reduce KO. Uncoupling was revealed because a second activity, a warfarin-resistant quinone reductase, was not present. In contrast, 293 cells expressing factor IX and this reductase activity showed much less inhibition of carboxylation. This activity therefore appears to cooperate with VKORC1 to accomplish full KO reduction. Cooperation during warfarin therapy would have significant consequences, as VKD proteins function in numerous physiologies in many tissues, but may be poorly carboxylated and dysfunctional if the second activity is not ubiquitously expressed similar to VKORC1.

VKOR paralog VKORC1L1 supports vitamin K-dependent protein carboxylation in vivo.

Vertebrates possess 2 proteins with vitamin K oxidoreductase (VKOR) activity: VKORC1, whose vitamin K reduction supports vitamin K-dependent (VKD) protein carboxylation, and VKORC1-like 1 (VKORC1L1), whose function is unknown. VKD proteins include liver-derived coagulation factors, and hemorrhaging and lethality were previously observed in mice lacking either VKORC1 or the γ-glutamyl carboxylase (GGCX) that modifies VKD proteins. Vkorc1-/- mice survived longer (1 week) than Ggcx-/- mice (midembryogenesis or birth), and we assessed whether VKORC1L1 could account for this difference. We found that Vkorc1-/-;Vkorc1l1-/- mice died at birth with severe hemorrhaging, indicating that VKORC1L1 supports carboxylation during the pre- and perinatal periods. Additional studies showed that only VKORC1 sustains hemostasis beyond P7. VKORC1 expression and VKOR activity increased during late embryogenesis and following birth, while VKORC1L1 expression was unchanged. At P0, most (>99%) VKOR activity was due to VKORC1. Prothrombin mRNA, protein, and carboxylation also increased during this period, as did mRNA levels of coagulation factors encoding genes F7, F9, and F10. VKORC1L1 levels in Vkorc1-/- mouse liver may therefore be insufficient for supporting carboxylation beyond day 7. In support of this conclusion, VKORC1L1 overexpression in liver rescued carboxylation and hemostasis in adult Vkorc1-/- mice. These findings establish that VKORC1L1 supports VKD protein carboxylation in vivo.

The vitamin K oxidoreductase is a multimer that efficiently reduces vitamin K epoxide to hydroquinone to allow vitamin K-dependent protein carboxylation.

The vitamin K oxidoreductase (VKORC1) recycles vitamin K to support the activation of vitamin K-dependent (VKD) proteins, which have diverse functions that include hemostasis and calcification. VKD proteins are activated by Glu carboxylation, which depends upon the oxygenation of vitamin K hydroquinone (KH2). The vitamin K epoxide (KO) product is recycled by two reactions, i.e. KO reduction to vitamin K quinone (K) and then to KH2, and recent studies have called into question whether VKORC1 reduces K to KH2. Analysis in insect cells lacking endogenous carboxylation components showed that r-VKORC1 reduces KO to efficiently drive carboxylation, indicating KH2 production. Direct detection of the vitamin K reaction products is confounded by KH2 oxidation, and we therefore developed a new assay that stabilized KH2 and allowed quantitation. Purified VKORC1 analyzed in this assay showed efficient KO to KH2 reduction. Studies in 293 cells expressing tagged r-VKORC1 revealed that VKORC1 is a multimer, most likely a dimer. A monomer can only perform one reaction, and a dimer is therefore interesting in explaining how VKORC1 accomplishes both reactions. An inactive mutant (VKORC1(C132A/C135A)) was dominant negative in heterodimers with wild type VKORC1, resulting in decreased KO reduction in cells and carboxylation in vitro. The results are significant regarding human VKORC1 mutations, as warfarin-resistant patients have mutant and wild type VKORC1 alleles. A VKORC1 dimer indicates a mixed population of homodimers and heterodimers that may have different functional properties, and VKORC1 reduction may therefore be more complex in these patients than appreciated previously.

Vitamin K oxygenation, glutamate carboxylation, and processivity: defining the three critical facets of catalysis by the vitamin K-dependent carboxylase.

The vitamin K-dependent carboxylase uses vitamin K oxygenation to drive carboxylation of multiple glutamates in vitamin K-dependent proteins, rendering them active in a variety of physiologies. Multiple carboxylations of proteins are required for their activity, and the carboxylase is processive, so that premature dissociation of proteins from the carboxylase does not occur. The carboxylase is unique, with no known homology to other enzyme families, and structural determinations have not been made, rendering an understanding of catalysis elusive. Although a model explaining the relationship of oxygenation to carboxylation had been developed, until recently almost nothing was known of the function of the carboxylase itself in catalysis. In the past decade, discovery and analysis of naturally occurring carboxylase mutants has led to identification of functionally relevant residues and domains. Further, identification of nonmammalian carboxylase orthologs has provided a basis for bioinformatic analysis to identify candidates for critical functional residues. Biochemical analysis of rationally chosen carboxylase mutants has led to breakthroughs in understanding vitamin K oxygenation, glutamate carboxylation, and maintenance of processivity by the carboxylase. Protein carboxylation has also been assessed in vivo, and the intracellular environment strongly affects carboxylase function. The carboxylase is an integral membrane protein, and topological analysis, coupled with biochemical determinations, suggests that interaction of the carboxylase with the membrane is an important facet of function. Carboxylase homologs, likely acquired by horizontal transfer, have been discovered in some bacteria, and functional analysis of these homologs has the potential to lead to the discovery of new roles of vitamin K in biology.

The vitamin K-dependent carboxylase generates γ-carboxylated glutamates by using CO2 to facilitate glutamate deprotonation in a concerted mechanism that drives catalysis.

The γ-glutamyl carboxylase converts Glu to carboxylated Glu (Gla) to activate a large number of vitamin K-dependent proteins with diverse functions, and this broad physiological impact makes it critical to understand the mechanism of carboxylation. Gla formation is thought to occur in two independent steps (i.e. Glu deprotonation to form a carbanion that then reacts with CO(2)), based on previous studies showing unresponsiveness of Glu deprotonation to CO(2). However, our recent studies on the kinetic properties of a variant enzyme (H160A) showing impaired Glu deprotonation prompted a reevaluation of this model. Glu deprotonation monitored by tritium release from the glutamyl γ-carbon was dependent upon CO(2), and a proportional increase in both tritium release and Gla formation occurred over a range of CO(2) concentrations. This discrepancy with the earlier studies using microsomes is probably due to the known accessibility of microsomal carboxylase to water, which reprotonates the carbanion. In contrast, tritium incorporation experiments with purified carboxylase showed very little carbanion reprotonation and consequently revealed the dependence of Glu deprotonation on CO(2). Cyanide stimulated Glu deprotonation and carbanion reprotonation to the same extent in wild type enzyme but not in the H160A variant. Glu deprotonation that depends upon CO(2) but that also occurs when water or cyanide are present strongly suggests a concerted mechanism facilitated by His-160 in which an electrophile accepts the negative charge on the developing carbanion. This revised mechanism provides important insight into how the carboxylase catalyzes the reaction by avoiding the formation of a high energy discrete carbanion.

Novel insight into the mechanism of the vitamin K oxidoreductase (VKOR): electron relay through Cys43 and Cys51 reduces VKOR to allow vitamin K reduction and facilitation of vitamin K-dependent protein carboxylation.

The vitamin K oxidoreductase (VKOR) reduces vitamin K to support the carboxylation and consequent activation of vitamin K-dependent proteins, but the mechanism of reduction is poorly understood. VKOR is an integral membrane protein that reduces vitamin K using membrane-embedded thiols (Cys-132 and Cys-135), which become oxidized with concomitant VKOR inactivation. VKOR is subsequently reactivated by an unknown redox protein that is currently thought to act directly on the Cys132-Cys135 residues. However, VKOR contains evolutionarily conserved Cys residues (Cys-43 and Cys-51) that reside in a loop outside of the membrane, raising the question of whether they mediate electron transfer from a redox protein to Cys-132/Cys-135. To assess a possible role, the activities of mutants with Ala substituted for Cys (C43A and C51A) were analyzed in intact membranes using reductants that were either membrane-permeable or -impermeable. Both reductants resulted in wild type VKOR reduction of vitamin K epoxide; however, the C43A and C51A mutants only showed activity with the membrane-permeant reductant. We obtained similar results when testing the ability of wild type and mutant VKORs to support carboxylation, using intact membranes from cells coexpressing VKOR and carboxylase. These results indicate a role for Cys-43 and Cys-51 in catalysis, suggesting a relay mechanism in which a redox protein transfers electrons to these loop residues, which in turn reduce the membrane-embedded Cys132-Cys135 disulfide bond to activate VKOR. The results have implications for the mechanism of warfarin resistance, the topology of VKOR in the membrane, and the interaction of VKOR with the carboxylase.

Mutations in the GGCX and ABCC6 genes in a family with pseudoxanthoma elasticum-like phenotypes.

A characteristic feature of classic pseudoxanthoma elasticum (PXE), an autosomal recessive disorder caused by mutations in the ABCC6 gene, is aberrant mineralization of connective tissues, particularly the elastic fibers. Here, we report a family with PXE-like cutaneous features in association with multiple coagulation factor deficiency, an autosomal recessive disorder associated with GGCX mutations. The proband and her sister, both with severe skin findings with extensive mineralization, were compound heterozygotes for missense mutations in the GGCX gene, which were shown to result in reduced gamma-glutamyl carboxylase activity and in undercarboxylation of matrix gla protein. The proband's mother and aunt, also manifesting with PXE-like skin changes, were heterozygous carriers of a missense mutation (p.V255M) in GGCX and a null mutation (p.R1141X) in the ABCC6 gene, suggesting digenic nature of their skin findings. Thus, reduced gamma-glutamyl carboxylase activity in individuals either compound heterozygous for a missense mutation in GGCX or with haploinsufficiency in GGCX in combination with heterozygosity for ABCC6 gene expression results in aberrant mineralization of skin leading to PXE-like phenotype. These findings expand the molecular basis of PXE-like phenotypes, and suggest a role for multiple genetic factors in pathologic tissue mineralization in general.

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.

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.

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.

2H, 13C, and 15N kinetic isotope effects on the reaction of the ammonia-rescued K258A mutant of aspartate aminotransferase.

Deuterium isotope effects at C2 of aspartate and heavy atom isotope effects at C2, C3, and the amino group of aspartate were determined for the reaction of the lysine-258 to alanine mutant of Escherichia coli rescued with exogenous ammonia. We were able to calculate an (15)N intrinsic isotope effect of 1.034. The intrinsic (13)C isotope effect at C3 is 1.0060, and the (13)C isotope effect at C2 is 1.0016. These isotope effects reveal that collapse of the carbinolamine (or gem-diamine) to give the final product is the rate-determining step in this system. Furthermore, these results indicate that lysine-258 is critical to the catalysis of the final breakdown to give product, and in fact this step is more strongly affected by mutation of lysine-258 than the deprotonation of the external aldimine.