A characteristic feature of classic 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 γ-glutamyl carboxylase activity and in under-carboxylation 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 γ-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.

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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. Adv. Nutr. 3: 135-148, 2012.

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.

Background: How the vitamin K oxidoreductase (VKORC1) supports vitamin K-dependent protein carboxylation is poorly understood. Results: VKORC1 multimers efficiently perform both reactions that reduce vitamin K, and the inactive monomer in wild type mutant heteromers suppresses reduction. Conclusion: VKORC1 fully reduces vitamin K required for carboxylation. Significance: Multimers are important in VKORC1 mechanism and wild type mutant heteromers impact patients with warfarin resistance.

No abstract

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 wildtype 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 wildtype 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 Km for vitamin K and caused only a 2-fold increase in the Km for Glu and 2-fold decrease in the activation of vitamin K epoxidation by Glu. The H160A Km for CO2 was 5-fold higher than wildtype enzyme. However, the kcat for H160A carboxylation was 8 to 9-fold lower than wildtype enzyme with all three substrates (i.e. Glu, CO2 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 orthologs (acquired by horizontal gene transfer), which has implications for how bacteria have adapted the carboxylase for novel functions.

A listing of 13 C, 15 N, and 18 O equilibrium isotope effects and fractionation factors for atoms in specific positions is provided. Empirical factors that can be used to adjust these fractionation factors for more complex structures are presented and discussed. While much work needs to be done to determine equilibrium isotope effects for these heavy atoms, the values tabulated here should be useful to anyone working with isotope effects involving these atoms. Résumé: On présente une liste des effets isotopiques d'équilibre et des facteurs de fractionnement du 13 C, du 15 N et du 18 O relatifs à des atomes dans des positions spécifiques. On présente et on discute des facteurs empiriques qui peuvent être utilisés pour ajuster ces facteurs de fractionnement dans les cas de structures plus complexes. Même si beaucoup de travail reste à faire pour déterminer les effets isotopiques d'équilibre pour ces atomes lourds, les valeurs rapportées ici devraient s'avérer utiles pour quiconque s'intéresse aux effets isotopiques impliquant ces atomes. Mots clés : effets isotopiques d'équilibre, facteurs de fractionnement, 13 C, 15 N et 18 O. [Traduit par la Rédaction] Rishavy and Cleland 977Substitution of a heavier isotope for a lighter one not only affects the rates of chemical reactions, but also changes equilibrium constants if the isotopic atom is more stiffly bonded in the reactant than in the product or vice versa. Since the equations for kinetic isotope effects include the equilibrium isotope effect in one of the numerator terms (1), it is important for anyone trying to interpret kinetic isotope effects to know these values.Equilibrium isotope effects are commonly expressed as the ratio of K eq for the light isotope to K eq for the heavy one. A leading superscript indicates the isotope involved. Thus, 13 K eq is K eq C-12 /K eq C-13 . 14 K eq , 15 K eq , and 18 K eq are equilibrium isotope effects caused by replacement of 12 C, 14 N, or 16 O by 14 C, 15 N, or 18 O, respectively. Values greater than unity show that the heavy isotope is enriched in the reactant, while values less than unity show enrichment in the product. The equilibrium isotope effect in the back reaction is the reciprocal of that in the forward reaction. For comparison of 14 C and 13 C equilibrium isotope effects (2), Can. J. Chem. 77: 967-977 (1999)