Vitamin K-dependent carboxylation. Mechanistic studies with 3-fluoroglutamate-containing substrates.
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ABSTRACT:
SUBMITTER: Vidal-Cros A
PROVIDER: S-EPMC1131203 | biostudies-other | 1990 Mar
REPOSITORIES: biostudies-other
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Project description:The peptide substrate commonly used in vitamin K-dependent carboxylation, Phe-Leu-Glu-Glu-Val, has been shown, by the use of high-voltage paper electrophoresis, to be degraded from the N-terminus by a microsomal leucine amino-peptidase. The replacement of phenylalanine with a N-t-butoxycarbonyl group resulted in a tetrapeptide substrate with a blocked N-terminus resistant to enzymic degradation. Vitamin K-dependent carboxylation of this non-degradable substrate gave a unique carboxylated product, which was separated from microsomal protein and unchanged substrate by using DEAE-Sephadex A25 as a final step. The carboxylated product was subsequently decarboxylated in 2HCl and analysed by using g.l.c. coupled to a mass spectrometer. This showed that only the first glutamic acid residue in the peptide substrate was carboxylated.
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Project description: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.
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Project description:The nickel-catalyzed carboxylation of organic halides or pseudohalides using carbon dioxide is an emerging method to prepare synthetically valuable carboxylic acids. Here, we report a detailed mechanistic investigation of these reactions using the carboxylation of aryl halides with (PPh3)2NiIICl2 as a model reaction. Our studies allow us to understand several general features of nickel-catalyzed carboxylation reactions. For example, we demonstrate that both a Lewis acid and halide source are beneficial for catalysis. To this end, we establish that heterogeneous Mn(0) and Zn(0) reductants are multifaceted reagents that generate noninnocent Mn(II) or Zn(II) Lewis acids upon oxidation. In a key result, a rare example of a well-defined nickel(I) aryl complex is isolated, and it is demonstrated that its reaction with carbon dioxide results in the formation of a carboxylic acid in high yield (after workup). The carbon dioxide insertion product undergoes rapid decomposition, which ca These three oxidation states correspond to the onbe circumvented by a ligand metathesis reaction with a halide source. Our studies have led to both a revised mechanism and the development of a broadly applicable strategy to improve reductive carboxylation reactions. A critical component of this strategy is that we have replaced the heterogeneous Mn(0) reductant typically used in catalysis with a well-defined homogeneous organic reductant. Through its use, we have increased the range of ancillary ligands, additives, and substrates that are compatible with the reaction. This has enabled us to perform reductive carboxylations at low catalyst loadings. Additionally, we demonstrate that reductive carboxylations of organic (pseudo)halides can be achieved in high yields in more practically useful, non-amide solvents. Our results describe a mechanistically guided strategy to improve reductive carboxylations through the use of a homogeneous organic reductant, which may be broadly translatable to a wide range of cross-electrophile coupling reactions.
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Project description:Periostin (PN, gene name POSTN) is an extracellular matrix protein that is up-regulated in bronchial epithelial cells and lung fibroblasts by TH-2 cytokines. Its paralog, TGF-?-induced protein (?ig-h3, gene name TGFBI), is also expressed in the lung and up-regulated in bronchial myofibroblasts by TGF-?. PN and ?ig-h3 contain fasciclin 1 modules that harbor putative recognition sequences for ?-glutamyl carboxylase and are annotated in UniProt as undergoing vitamin K-dependent ?-carboxylation of multiple glutamic acid residues. ?-carboxylation profoundly alters activities of other proteins subject to the modification, e.g., blood coagulation factors, and would be expected to alter the structure and function of PN and ?ig-h3. To analyze for the presence of ?-carboxylation, proteins extracted from fibrotic lung were reacted with monoclonal antibodies specific for PN, ?ig-h3, or modification with ?-carboxyglutamic acid (Gla). In Western blots of 1-dimensional gels, bands stained with anti-PN or -?ig-h3 did not match those stained with anti-Gla. In 2-dimensional gels, anti-PN-positive spots had pIs of 7.0 to >8, as expected for the unmodified protein, and there was no overlap between anti-PN-positive and anti-Gla-positive spots. Recombinant PN and blood coagulation factor VII were produced in HEK293 cells that had been transfected with vitamin K 2, 3-epoxide reductase C1 to optimize ?-carboxylation. Recombinant PN secreted from these cells did not react with anti-Gla antibody and had pIs similar to that found in extracts of fibrotic lung whereas secreted factor VII reacted strongly with anti-Gla antibody. Over 67% coverage of recombinant PN was achieved by mass spectrometry, including peptides with 19 of the 24 glutamates considered targets of ?-carboxylation, but analysis revealed no modification. Over 86% sequence coverage and three modified glutamic acid residues were identified in recombinant fVII. These data indicate that PN and ?ig-h3 are not subject to vitamin K-dependent ?-carboxylation.
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Project description:Haloalkane dehalogenases are enzymes that catalyze the cleavage of carbon-halogen bonds in halogenated compounds. They serve as model enzymes for studying structure-function relationships of >100.000 members of the α/β-hydrolase superfamily. Detailed kinetic analysis of their reaction is crucial for understanding the reaction mechanism and developing novel concepts in protein engineering. Fluorescent substrates, which change their fluorescence properties during a catalytic cycle, may serve as attractive molecular probes for studying the mechanism of enzyme catalysis. In this work, we present the development of the first fluorescent substrates for this enzyme family based on coumarin and BODIPY chromophores. Steady-state and pre-steady-state kinetics with two of the most active haloalkane dehalogenases, DmmA and LinB, revealed that both fluorescent substrates provided specificity constant two orders of magnitude higher (0.14-12.6 μM-1 s-1) than previously reported representative substrates for the haloalkane dehalogenase family (0.00005-0.014 μM-1 s-1). Stopped-flow fluorescence/FRET analysis enabled for the first time monitoring of all individual reaction steps within a single experiment: (i) substrate binding, (ii-iii) two subsequent chemical steps and (iv) product release. The newly introduced fluorescent molecules are potent probes for fast steady-state kinetic profiling. In combination with rapid mixing techniques, they provide highly valuable information about individual kinetic steps and mechanism of haloalkane dehalogenases. Additionally, these molecules offer high specificity and efficiency for protein labeling and can serve as probes for studying protein hydration and dynamics as well as potential markers for cell imaging.
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Project description:Recent research has shown that rat surfactant apoproteins (26-38 kDa) are vitamin K-dependent [Rannels, Gallaher, Wallin & Rannels (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5952-5956]. We have investigated the effect of the vitamin K antagonist warfarin on this family of apoproteins in surfactant from dog lung. Our data suggest that warfarin does not interfere with synthesis and secretion of these proteins into dog lung surfactant. Abnormal surfactant apoproteins, produced in response to warfarin treatment of the dog, were also not found in lung surfactant. 4-Carboxyglutamic acid analysis of purified dog apoproteins also failed to detect the vitamin K-modification. When vitamin K-dependent 14C labelling of precursors of vitamin K-dependent proteins was carried out, fluorography of these precursors, when electrophoresed into SDS/polyacrylamide gels, revealed 14C-labelled proteins of apparent molecular mass 74, 46, 42, 34, 31 and 23 kDa. Antibodies produced against purified dog surfactant apoproteins recognized precursors of the surfactant apoproteins in lung microsomes but did not recognize any 14C-labelled carboxylase substrates. These precursors appeared on immunoblots with apparent molecular mass 29, 32, 33 and 50 kDa. Our data suggest that there are significant differences between this class of surfactant apoproteins in the rat and the dog.
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Project description: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.
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Project description:Passage of a Triton X-100-solubilized microsomal systems in vitro that are used to study these reactions is the warfarin-sensitive NAD(P)H dehydrogenase.
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Project description: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 gamma-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- N-ethyl maleimide-modified human carboxylase revealed 1.8-2.3 reactive residues and a specific activity of 7 x 10(8) cpm/hr 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(m)s for a glutamyl substrate 6- to 8-fold. Retention of some activity indicates a mechanism for enhancing cysteine/serine 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.
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