Vitamin K-dependent carboxylation. Mechanistic studies with 3-fluoroglutamate-containing substrates

Vidal-Cros, Anne; Gaudry, Michel; Marquet, Andrée

doi:10.1042/bj2660749

Cited by 13 publications

(8 citation statements)

References 18 publications

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“…Possibly there will be no discrimination between the two diastereomers since C-3 is converted into a methyl group. None of the fluoroglutamates gave rise to fluoride elimination in the complete assay, indicating the absence of any carbanion intermediate [35]. …”

Section: Discussionmentioning

confidence: 99%

Glutamate mutase from Clostridium cochlearium

Leutbecher

Böcher

Linder

et al. 1992

European Journal of Biochemistry

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Both components, E and S, of the adenosylcobalamin-(coenzyme B1 ,)-dependent glutamate mutase from Clostridium cochlearium were purified. Component S (16 kDa) must be added to component E to obtain activity, although the latter contains substoichiometric amounts of component S besides the major 50-kDa subunit. The enzyme proved to be very similar to that of C . tetanomorphum Substitution of the migrating hydrogen at C-4 of glutamate by fluorine yielded the potent competitive inhibitor (2S,4S)-4-fluoroglutamate (Ki = 70 pM). (2R,3RS)-3-Fluoroglutamate (KI = 600 pM) was also inhibitory. The competitive inhibition by 2-methyleneglutarate (Ki = 400 pM) and (59-3-methylitaconate (Ki = 100 pM) but not by (RS)-2-methylglutarate suggested the transient formation of an sp2 center during catalysis. However, the presence of an N-terminal pyruvoyl residue was excluded and no evidence for the participation of another electrophilic center in the reaction was obtained.Glutamate mutase catalyses the reversible rearrangement of (S)-glutamate to (2S,3S)-3-methylaspartate, the first step in the fermentation of glutamate by Clostridium tetanomorphum [l] and related clostridia [2]. The elucidation of the coenzyme of this reaction as an adenosyl derivative of pseudovitamin B was a landmark in biochemistry [3] leading to the discovery of the family of coenzyme-B, 2-dependent enzymes (for reviews see 14-91). Until now, the role of coenzyme B12 in those rearrangements has not been clear.In general, homolytic cleavage of the cobalt-carbon bond is agreed to be the first step in the catalysis of adenosylcobalamin-dependent enzymes. The resulting S-deoxyadenosyl radical should abstract a hydrogen atom yielding the substrate radical which rearranges to the product radical. Redonation of a hydrogen leads to the product and regenerates the . Such a mechanism is unlikely in the glutamate mutase reaction due to the lack of a double bond at the migrating carbon. Indeed, chemical modelling showed that glutamate derivatives only rearranged if the amino acid was converted to a ketimine [16] suggesting the participation of an electrophilic center in the reaction. Pyridoxal phosphate, however, the most likely prosthetic group, was not detected [17].Glutamate mutase from C. tetanomorphum is composcd of two components: E (128 kDa) and S (17 kDa). Due to the instability of component E, only component S was purified to homogeneity [7, 181. This paper describes the purification of components E and S of the closely related C. cochlarium to high purity and investigations on the mechanistic questions

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Section: Discussionmentioning

confidence: 99%

Glutamate mutase from Clostridium cochlearium

Leutbecher

Böcher

Linder

et al. 1992

European Journal of Biochemistry

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“…Rev., 2012, 41, 2135-2171 2155 of glutamic acid using the tripeptide (Boc-Xaa-Glu-[ 3 H]Val). 173 The authors hypothesized that since b-fluoro carbanions are prone to fluoride elimination, they should be able to distinguish a radical-mediated mechanism from ion-mediated mechanism by identifying the final product (Fig. 29).…”

Section: Analogues Of Polar and Charged Amino Acids 41 General Proper...mentioning

confidence: 99%

Fluorinated amino acids: compatibility with native protein structures and effects on protein–protein interactions

et al. 2012

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Fluorinated analogues of the canonical α-L-amino acids have gained widespread attention as building blocks that may endow peptides and proteins with advantageous biophysical, chemical and biological properties. This critical review covers the literature dealing with investigations of peptides and proteins containing fluorinated analogues of the canonical amino acids published over the course of the past decade including the late nineties. It focuses on side-chain fluorinated amino acids, the carbon backbone of which is identical to their natural analogues. Each class of amino acids--aliphatic, aromatic, charged and polar as well as proline--is presented in a separate section. General effects of fluorine on essential properties such as hydrophobicity, acidity/basicity and conformation of the specific side chains and the impact of these altered properties on stability, folding kinetics and activity of peptides and proteins are discussed (245 references).

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“…Use of short Glu-containing peptide substrate analogs established that Glu underwent deprotonation at the g carbon, leading to reaction of CO 2 at that site to form Gla ( Fig. 2A), as opposed to a mechanism involving abstraction of a hydrogen radical (1,2). g Carbon deprotonation would be extremely difficult, however, requiring action of a base far stronger than any possible enzyme active site residue.…”

Section: Introductionmentioning

confidence: 99%

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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Vitamin K-dependent carboxylation. Mechanistic studies with 3-fluoroglutamate-containing substrates

Cited by 13 publications

References 18 publications

Glutamate mutase from Clostridium cochlearium

Glutamate mutase from Clostridium cochlearium

Fluorinated amino acids: compatibility with native protein structures and effects on protein–protein interactions

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

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