Congenital Deficiency of All Vitamin K-Dependent Blood Coagulation Factors Due to a Defective Vitamin K-Dependent Carboxylase in Devon Rex Cats

Soute, Berry A.M.; Ulrich, Magda M. W.; Watson, A. D. J.; Maddison, Jill; Ebberink, R.H.M.; Vermeer, Cees

doi:10.1055/s-0038-1646311

Cited by 72 publications

(51 citation statements)

References 10 publications

(13 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The K m of the wild-type FLAG-CBX for vitamin KH 2 , determined at 3.6 mM FLEEL and 16 M proFIX18, is 73.7 Ϯ 14.0 M (Table VI). This is within the range of K m values of 10 -100 M previously reported (8,32,33). The four mutants, CBX234/235, CBX359/ 360/361, CBX406/408, and CBX513/515, have similar K m values to that of the wild-type enzyme.…”

Section: Flag-cbxsupporting

confidence: 66%

“…When 5 M proPT28 is used as a substrate, wild-type FLAG-CBX has a K m for vitamin KH 2 of 4.0 Ϯ 0.4 M, an 18-fold lower value than that observed using FLEEL. It has been reported that the K m for vitamin KH 2 is much lower in the presence of a propeptide-containing substrate than a substrate lacking the carboxylation recognition site (8,33). CBX234/235 and CBX406/408 showed similar decreases in K m for vitamin K in the presence of proPT28.…”

Section: Flag-cbxmentioning

confidence: 58%

See 1 more Smart Citation

Profactor IX Propeptide and Glutamate Substrate Binding Sites on the Vitamin K-dependent Carboxylase Identified by Site-directed Mutagenesis

Sugiura

Furie

Walsh

et al. 1996

Journal of Biological Chemistry

View full text Add to dashboard Cite

The vitamin K-dependent carboxylase, a constituent of the endoplasmic reticulum membrane, catalyzes the conversion of reduced vitamin K to vitamin K epoxide and the concomitant conversion of glutamic acid to ␥-carboxyglutamic acid. To study structure-function relationships in the enzyme, seventeen clusters of charged residues of the bovine ␥-glutamyl carboxylase were substituted with alanines using site-specific mutagenesis. Wild-type and mutant carboxylase species were expressed in Chinese hamster ovary cells with an immunodetectable octapeptide inserted at their amino-terminal ends. Out of 17 mutant carboxylase species that contain a total of 41 charged residue to alanine substitutions, K217A/K218A (CBX217/218), R234A/H235A (CBX234/235), R359A/H360A/K361A (CBX359/360/361), R406A/H408A (CBX406/408), and R513A/K515A (CBX513/ 515) had impaired carboxylase activity compared with the wild-type enzyme. The vitamin K epoxidase activities of these mutants were reduced in parallel with the carboxylase activities. CBX217/218 appears to be inactive. High propeptide concentrations were required for stimulation of carboxylation of FLEEL by CBX234/235, CBX406/408, and CBX513/515, suggesting defects in the propeptide binding site. CBX359/360/361 showed normal affinity for the propeptide, FLEEL, proPT28, and vitamin K hydroquinone but exhibited a low catalytic rate for carboxylation. These results suggest that residue 217, residue 218, or both are either critical for catalysis or for maintaining the structure of a catalytically active enzyme. Regions around residues 234, 406, and 513 define in part the propeptide binding site, while the regions around residue 359 are involved in catalysis.Vitamin K-dependent ␥-glutamyl carboxylase is a membrane-associated endoplasmic reticulum resident enzyme that catalyzes the posttranslational conversion of specific glutamic acids of the vitamin K-dependent proteins to Gla. The vitamin K-dependent blood-clotting and regulatory proteins have clusters of 9 -12 Gla residues near their N termini. A Ca 2ϩ

show abstract

Section: Flag-cbxsupporting

confidence: 66%

Section: Flag-cbxmentioning

confidence: 58%

Profactor IX Propeptide and Glutamate Substrate Binding Sites on the Vitamin K-dependent Carboxylase Identified by Site-directed Mutagenesis

Sugiura

Furie

Walsh

et al. 1996

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Both mutants exhibited an increased K m for the glutamyl substrate, and Glu binding has been shown to increase carboxylase affinity for KH 2 (15,16), raising the possibility that Cys-99 and Cys-450 both are required to coordinate KH 2 in a substrate-dependent manner. The two thiols must be in close proximity to KH 2 because it protected them from NEM inactivation (Table 1).…”

Section: Discussionmentioning

confidence: 99%

“…The KH 2 preincubation was performed in the absence of a carboxylatable substrate to prevent vitamin K turnover and consequent exposure of the vitamin K protected site to NEM. Recent studies suggest that the carboxylatable substrate may affect KH 2 binding (15,16), and its absence may explain why only 62-64% protection of activity was observed at the concentration of KH 2 used (350 M). Higher concentrations also were attempted but led to inhibition of activity, as reported by others (17).…”

Section: Nem Inhibition Of Carboxylation and Epoxidation And Protectimentioning

confidence: 99%

Identification of the vitamin K-dependent carboxylase active site: Cys-99 and Cys-450 are required for both epoxidation and carboxylation

Pudota

Miyagi

Hallgren

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

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...

show abstract

“…The peptides, synthesized in a bacterial expression system, consist of the wild-type or mutant propeptide covalently linked to the Gla domain of F.IX. Studies by a number of groups (4,14,17,18) have shown that attaching the propeptide to the substrate lowers the apparent K m of reduced vitamin K for the carboxylase. Thus, for example, when the pentapeptide FLEEL is the substrate, the apparent K m for reduced vitamin K is ‫ف‬ 100 M, but a 59-amino acid sequence spanning the region from Ϫ18 to ϩ41 of the human F.IX protein has a K m app 100-fold lower ‫ف(‬ 1 M; reference 18).…”

Section: Discussionmentioning

confidence: 99%

A mutation in the propeptide of Factor IX leads to warfarin sensitivity by a novel mechanism.

Chu¹,

Wu²,

Stanley³

et al. 1996

J. Clin. Invest.

103

View full text Add to dashboard Cite

The propeptide sequences of the vitamin K-dependent clotting factors serve as a recognition site for the enzyme ␥ -glutamylcarboxylase, which catalyzes the carboxylation of glutamic acid residues at the NH 2 terminus of the mature protein. We describe a mutation in the propeptide of Factor IX that results in warfarin sensitivity because of reduced affinity of the carboxylase for the Factor IX precursor. The proband has a Factor IX activity level of Ͼ 100% off warfarin and Ͻ 1% on warfarin, at a point where other vitamin K-dependent factors were at 30-40% activity levels. Direct sequence analysis of amplified genomic DNA from all eight exons and exon-intron junctions showed a single guanosine → adenosine transition at nucleotide 6346 resulting in an alanine to threonine change at residue Ϫ 10 in the propeptide. To define the mechanism by which the mutation resulted in warfarin sensitivity, we analyzed wild-type and mutant recombinant peptides in an in vitro carboxylation reaction. The peptides that were analyzed included the wild-type sequence, the Ala-10 → Thr sequence, and Ala-10 → Gly, a substitution based on the sequence in bone ␥ -carboxyglutamic acid protein. Measurement of CO 2 incorporation at a range of peptide concentrations yielded a V max of 343 cpm/min/reaction for the wild-type peptide, and V max values of 638 and 726 for A-10T and A-10G respectively, a difference of only twofold. The K m values, on the other hand, showed a 33-fold difference between wild-type and the variants, with a value of 0.29 M for wild-type, and 10.9 and 9.50 M, respectively, for A-10T and A-10G. Similar kinetic experiments showed no substantial differences between wild-type and mutant peptides in kinetic parameters of the carboxylase-peptide complexes for reduced vitamin K. We conclude that the major defect resulting from the Factor IX Ala-10 → Thr mutation is a reduction in affinity of the carboxylase for the mutant propeptide. These studies delineate a novel mechanism for warfarin sensitivity. In addition, the data may also explain the observation that bone Gla protein is more sensitive to warfarin than the coagulation proteins. ( J. Clin. Invest. 1996. 98:1619-1625.)

show abstract

Congenital Deficiency of All Vitamin K-Dependent Blood Coagulation Factors Due to a Defective Vitamin K-Dependent Carboxylase in Devon Rex Cats

Cited by 72 publications

References 10 publications

Profactor IX Propeptide and Glutamate Substrate Binding Sites on the Vitamin K-dependent Carboxylase Identified by Site-directed Mutagenesis

Profactor IX Propeptide and Glutamate Substrate Binding Sites on the Vitamin K-dependent Carboxylase Identified by Site-directed Mutagenesis

Identification of the vitamin K-dependent carboxylase active site: Cys-99 and Cys-450 are required for both epoxidation and carboxylation

A mutation in the propeptide of Factor IX leads to warfarin sensitivity by a novel mechanism.

Contact Info

Product

Resources

About