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 vitamin K-dependent carboxylase modifies and renders active vitamin K-dependent proteins involved in hemostasis, cell growth control, and calcium homeostasis. Using a novel mechanism, the carboxylase transduces the free energy of vitamin K hydroquinone (KH 2) oxygenation to convert glutamate into a carbanion intermediate, which subsequently attacks CO 2, generating the ␥-carboxylated glutamate product. How the carboxylase effects this conversion is poorly understood because the active site has not been identified. Dowd and colleagues [Dowd, P., Hershline, R., Ham, S. W. & Naganathan, S. (1995) Science 269, 1684 -1691] have proposed that a weak base (cysteine) produces a strong base (oxygenated KH 2) capable of generating the carbanion. To define the active site and test this model, we identified the amino acids that participate in these reactions. N-ethyl maleimide inhibited epoxidation and carboxylation, and both activities were equally protected by KH 2 preincubation. Amino acid analysis of 14 C-Nethyl maleimide-modified human carboxylase revealed 1.8 -2.3 reactive residues and a specific activity of 7 ؋ 10 8 cpm͞hr per mg. Tryptic digestion and liquid chromatography electrospray mass spectrometry identified Cys-99 and Cys-450 as active site residues. Mutation to serine reduced both epoxidation and carboxylation, to 0.2% (Cys-99) or 1% (Cys-450), and increased the K ms for a glutamyl substrate 6-to 8-fold. Retention of some activity indicates a mechanism for enhancing cysteine͞serine nucleophilicity, a property shared by many active site thiol enzymes. These studies, which represent a breakthrough in defining the carboxylase active site, suggest a revised model in which the glutamyl substrate indirectly coordinates at least one thiol, forming a catalytic complex that ionizes a thiol to initiate KH 2 oxygenation. T he vitamin K-dependent (VKD) carboxylase is an integral membrane enzyme required for the biological activity of proteins involved in hemostasis (prothrombin, factor X, factor VII, factor IX, protein S, protein C, protein Z), calcium homeostasis (bone gla protein and matrix gla protein), cell growth control (gas 6), and possibly signal transduction (PRGP-1 and PRGP-2) (1, 2). Carboxylation is effected via a homologous Ϸ18-aa sequence in VKD proteins, usually an N-terminal propeptide, which the carboxylase binds with high affinity. Propeptide binding of VKD proteins to the carboxylase leads to the conversion of clusters of glutamyl (Glu) residues to ␥-carboxylated glutamyl (or Gla) residues, in a region adjacent to the propeptide called the Gla domain. This domain serves as a calcium-dependent membrane-binding module for the attached VKD proteins, for example to effect blood coagulation on cell surfaces. Carboxylation requires a continual supply of the reduced form of the vitamin K cofactor, vitamin K hydroquinone (KH 2 ), and when KH 2 is limiting undercarboxylated, inactive VKD proteins are produced. Consequently, understanding the mechanism of carboxylation has important medical ramifications as...

Vitamin K-dependent (VKD) proteins require modification by the VKD-␥-glutamyl carboxylase, an enzyme that converts clusters of glus to glas in a reaction that requires vitamin K hydroquinone, for their activity. We have discovered that the carboxylase also carboxylates itself in a reaction dependent on vitamin K. When pure human recombinant carboxylase was incubated in vitro with 14 CO 2 and then analyzed after SDS͞PAGE, a radiolabeled band corresponding to the size of the carboxylase was observed. Subsequent gla analysis of in vitro-modified carboxylase by base hydrolysis and HPLC showed that all of the radioactivity could be attributed to gla residues. Quantitation of gla, asp, and glu residues indicated 3 mol gla͞mol carboxylase. Radiolabeled gla was acid-labile, confirming its identity, and was not observed if vitamin K was not included in the in vitro reaction. Carboxylase carboxylation also was detected in baculovirus-(carboxylase)-infected insect cells but not in mock-infected insect cells, which do not express endogenous VKD proteins or carboxylase. Finally, we showed that the carboxylase was carboxylated in vivo. Carboxylase was purified from recombinant carboxylase BHK cells cultured in the presence or absence of vitamin K and analyzed for gla residues. Carboxylation of the carboxylase only was observed with carboxylase isolated from BHK cells cultured in vitamin K, and 3 mol gla͞mol carboxylase were detected. Analyses of carboxylase and factor IX carboxylation in vitro suggest a possible role for carboxylase carboxylation in factor IX turnover, and in vivo studies suggest a potential role in carboxylase stability. The discovery of carboxylase carboxylation has broad implications for the mechanism of VKD protein carboxylation and Warfarin-based anti-coagulant therapies that need to be considered both retrospectively and in the future.Vitamin K-dependent (VKD) proteins undergo an unusual posttranslational modification required for their biological activity, namely the carboxylation of clusters of glu residues to ␥-glutamyl glus, or glas, in a region of the VKD proteins called the gla domain (1, 2). Carboxylation of the VKD proteins effects their Ca 2ϩ -mediated interaction with phospholipid bilayers and is carried out by an integral membrane endoplasmic reticulum enzyme, the VKD-␥-glutamyl carboxylase. The carboxylase modifies VKD substrates by using CO 2 , O 2 , and vitamin K hydroquinone as cofactors, and the carboxylase is also an epoxidase, converting the vitamin K hydroquinone to vitamin K 2,3-epoxide. Subsequent regeneration of the vitamin K hydroquinone cofactor is carried out by a reductase that has been characterized but not yet identified (3, 4). Carboxylation of the gla domain involves the modification of multiple glus, ranging from 3 to 12 for the different VKD proteins. This multiplicity raises the question of whether the carboxylase is a processive enzyme, i.e., effecting all modifications as a result of a single binding event. When purified bovine liver carboxylase was coincubated...

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 (KH2), leading to KH2 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 KH2. [ 14 C]NEM modification of C99A͞C450A 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.

The vitamin K-dependent (VKD) carboxylase converts clusters of Glu residues to ␥-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 The vitamin K-dependent (VKD) 1 or ␥-carboxylase converts Glus to ␥-carboxylated Glus (Glas) in VKD proteins as they transit through the endoplasmic reticulum (1, 2). Most of the VKD proteins are secreted out of the cell, and carboxylation of their Gla domain confers the ability to bind phospholipid bilayers, where these proteins exert their effects. Carboxylation is thus required for the biological activity of VKD proteins, which function in hemostasis, calcium homeostasis, and growth control. In addition, a novel subset of mammalian VKD proteins with potential functions in signal transduction has recently been identified by sequence homology (3, 4). Unlike the other VKD proteins, these proteins apparently have a single-pass transmembrane domain with the extracellular domain containing the predicted carboxylated region. Inhibition of VKD protein activities forms the basis of anticoagulant therapies with warfarin and coumadin, in which the carboxylation of hemostatic VKD proteins, as well as the other VKD proteins, is reduced by limiting the supply of vitamin K cofactor to the carboxylase.Although the carboxylase was first identified in mammals, carboxylase homologs and activity have been found in fish, the fish-hunting cone snails of the genus Conus, and the fruit fly Drosophila (5-9). All chordates appear to contain the hemostatic VKD proteins (10). The known VKD proteins of Conus, however, have a distinct function where the VKD proteins are neurotoxic venom peptides (11)(12)(13)(14)(15). VKD proteins have not yet been isolated in Drosophila, and so the function of carboxylation in the fruit fly is not currently known.The carboxylase modifies VKD proteins by using O 2 and vitamin K hydroquinone (KH 2 ) to abstract the ␥-hydrogen of glutamyl residues to form a carbanion intermediate, which then incorporates CO 2 via nucleophilic attack to form the Gla (1, 2). During each Glu to Gla conversion, one molecule of KH 2 is oxidized to vitamin K epoxide, and the carboxylase is also an epoxidase. Insights into the molecular mechanism for this reaction have only recently been revealed. Early studies with thiol-specific inhibitors implicated Cys residues as part of the carboxylase active site (16). Chemical modeling based on those studies led to a proposed base strength amplification mechanism where a weak base (thiolate) initiates KH 2 oxygenation to generate a strong base that can abstract the