Mechanism of cyanide inhibition of the blood-clotting, vitamin K-dependent carboxylase.

Dowd, Paul; Ham, Seung Wook

doi:10.1073/pnas.88.23.10583

Cited by 6 publications

(2 citation statements)

References 17 publications

(10 reference statements)

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“…Tritium incorporation into the FLEEL peptide occurred specifically in a Glu residue, as shown by peptide hydrolysis followed by amino acid separation and quantitation using HPLC and scintillation counting (data not shown). Cyanide also stimulated vitamin K epoxidation, and the response was similar to that previously reported (43).…”

Section: Table 2 Carboxylase In Insect Cells Co-expressing Factor IX supporting

confidence: 78%

The Vitamin K-dependent Carboxylase Generates γ-Carboxylated Glutamates by Using CO2 to Facilitate Glutamate Deprotonation in a Concerted Mechanism That Drives Catalysis

Rishavy

Hallgren

Berkner

2011

Journal of Biological Chemistry

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The ␥-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 ␥-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.

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Section: Table 2 Carboxylase In Insect Cells Co-expressing Factor IX supporting

confidence: 78%

The Vitamin K-dependent Carboxylase Generates γ-Carboxylated Glutamates by Using CO2 to Facilitate Glutamate Deprotonation in a Concerted Mechanism That Drives Catalysis

Rishavy

Hallgren

Berkner

2011

Journal of Biological Chemistry

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“…In contrast, with microsomal carboxylase, the CO 2 site is accessible to water, so that concerted reprotonation by water can support Glu deprotonation. This interpretation is supported by the observation that cyanide, a known competitive inhibitor of CO 2 (53), can support Glu deprotonation in affinity-purified carboxylase (50), acting in a manner similar to water in microsomal carboxylase.…”

Section: Catalysis Of Vitamin K Oxygenation By the Carboxylasementioning

confidence: 80%

Vitamin K Oxygenation, Glutamate Carboxylation, and Processivity: Defining the Three Critical Facets of Catalysis by the Vitamin K–Dependent Carboxylase

Rishavy

Berkner

2012

Advances in Nutrition

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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.

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Walder¹,

Hucho²,

Döscher³

et al. 1992

Chemie in nserer Zeit

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Molmassenberechnung aus der chemischen Formel) (t, ng, L, hL, gal., Torr, mbar usw.). Volumen und Dichte der Stoffportion. Ordnungszahlen, Anzahl Atome eines Elementes, Masse einer Atomart in der Stoffportion, Massenanteil eines Elernentes, Elementmengenverhaltnisse (Elementaranalyse) -Relne Substanz als Bestandtell elner MISCHPHASE: Mengen-, Massen-, Volumen konzentratlon, Molalltat Mengen-, Massen-, Volumenanteil Masse, Volumen, Dichte der Gesamtphase Masse, Volumen, Dlchte, Stoffrnenge des Phasenrestes. -M n e n rnit dem IDEALEN GASGESETZ fur die reine Substanz, die Mischphase oder den Phasenrest; Berucksichtigung eines Realfaktors. -Anwendung der StBchlometrle auf alle Komponenten elner REAKTIONSGLEICHUNG (Auswertung von MaBanalysen). -SERIENRECHNUNGEN: Abspelchern aller Daten zu elner Problemstellung in einer Formeldatel, be1 Bedarf wleder ladbar. Das Programm STC)CHIOMEIWE ist auch Bestandteil der VISPER-32 Programmsammlung HOCHSCHULPAKET. Fordern Sic die ausfuhrlkhc Produktinformation an oder bestellen Sie gleich die Demo-Version fur DM 90,-(inkl. Handbuch, wird beim Kauf des Gesamtprogramms angerechnct). VISPER-32 Programme sind erhiiltlich fur: -IBM PC/XT/AT und PSD sowie zu diesen kompatible Rechner -Olivetti M24,MB -Apple Macintosh -Atari ST -K W S S A M Chrmie in unserer Zeit / 26. Jahrg. 1992 i Nr. 2

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Mechanism of cyanide inhibition of the blood-clotting, vitamin K-dependent carboxylase.

Cited by 6 publications

References 17 publications

The Vitamin K-dependent Carboxylase Generates γ-Carboxylated Glutamates by Using CO2 to Facilitate Glutamate Deprotonation in a Concerted Mechanism That Drives Catalysis

The Vitamin K-dependent Carboxylase Generates γ-Carboxylated Glutamates by Using CO2 to Facilitate Glutamate Deprotonation in a Concerted Mechanism That Drives Catalysis

Vitamin K Oxygenation, Glutamate Carboxylation, and Processivity: Defining the Three Critical Facets of Catalysis by the Vitamin K–Dependent Carboxylase

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