Post-translational activation introduces a free radical into pyruvate formate-lyase.

Knappe, J; Neugebauer, Franz A.; Blaschkowski, Hans P.; Ganzler, Melita

doi:10.1073/pnas.81.5.1332

Cited by 232 publications

(214 citation statements)

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“…In the latter system 5'-deoxyadenosine is generated and a free radical is introduced into the lyase [40]. However, neither event could be demonstrated yet in the dehydratase systems.…”

Section: Discussionmentioning

confidence: 99%

Purification of 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans. An iron-sulfur protein

1987

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The (R)‐2‐hydroxyglutaryl‐CoA dehydratase system from Acidaminococcus fermentans was separated by chromatography of cell‐free extracts on Q‐Sepharose into two components, an activator and the actual dehydratase. The latter enzyme was further purified to homogeneity by chromatography on blue‐Sepharose. It is an iron‐sulfur protein (Mr 210000) consisting of two different polypeptides (α, Mr 55000, and β, Mr 42000) in an α2β2 structure with probably two [4Fe‐4S] centers. After activation this purified enzyme catalysed the dehydration of (R)‐2‐hydroxyglutarate only in the presence of acetyl‐CoA and glutaconate CoA‐transferase, demonstrating that the thiol ester and not the free acid is the substrate of the dehydration. The result led to a modification of the hydroxyglutarate pathway of glutamate fermentation. The activation of the dehydratase by the flow‐through from Q‐Sepharose concentrated by ultrafiltration required NADH, MgCl2, ATP and strict anaerobic conditions. This fraction was designated as A°. Later when the concentration was performed by chromatography on phenyl‐Sepharose, an NADH‐independent form of the activator, designated as A*, was obtained. This enzyme, which required only ATP for activation of the dehydratase, was purified further by affinity chromatography on ATP‐agarose. It contains neither iron nor inorganic sulfur. A*, as well as the activated dehydratase, were irreversibly inactivated by exposure to air within less than 15 min. The activated dehydratase but not A* was also inactivated by 1 mM hydroxylamine or by 0.1 mM 2,4‐dinitrophenol. The (R)‐2‐hydroxyglutaryl‐CoA dehydratase system is closely related the that of (R)‐lactoyl‐CoA dehydratase from Clostridium propionicum as described by R. D. Kuchta and R. H. Abeles [(1985) J. Biol. Chem. 260, 13181–13189].

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“…In the latter system 5'-deoxyadenosine is generated and a free radical is introduced into the lyase [40]. However, neither event could be demonstrated yet in the dehydratase systems.…”

Section: Discussionmentioning

confidence: 99%

Purification of 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans. An iron-sulfur protein

1987

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“…At the post-translational level, pyruvate formate-lyase is interconverted by specific converter enzymes between a fully inactive form (Ei) and an active form (E,) that contains a persistent organic free radical [3]. Pyruvate is nonoxidatively processed to acetyl-CoA by a two-step reaction pattern with an intermediary acetyl-enzyme:…”

Section: Transcription Of the Structural Gene (~F L )mentioning

confidence: 99%

“…Pure radical enzyme (about 200 Ujmg) was obtained from homogeneous preparations of the non-radical form (Ei) which was carried through the conversion process with purified activating enzyme [3,71. To transfer enzyme samples between H 2 0 and DzO buffer, gel-filtration on Sephadex G-25 columns was used.…”

Section: Materidrmentioning

confidence: 99%

The free radical of pyruvate formate‐lyase

Unkrig

Neugebauer

Knappe

1989

European Journal of Biochemistry

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The first-derivative EPR spectrum of the active form of Escherichiu coli pyruvate formate-lyase shows an asymmetric doublet with partially resolved hyperfine splittings (g = 2.0037). Isotope substitution studies demonstrated couplings of a carbon-centered unpaired electron to a solvent-exchangeable proton (a = 1.5 mT) and to further hydrogen nuclei (a = 0.36 and 0.57 mT). By selective incorporation of unlabelled tyrosine into 2H-labelled enzyme protein, a tyrosyl radical structure has been ruled out. Circumstantial evidence indicates that the organic free radical, which also displays an ultraviolet absorption signal at 365 nm, is located on a standard amino acid residue of the polypeptide chain. EPR signal quantification found a stoichiometry of I spin per active site.The formate analogue hypophosphite has been characterized as a specific k,,, inhibitor of pyruvate formatelyase which destroys the enzyme radical. Protein-linked 1 -hydroxyethylphosphonate was previously described as the dead-end product after reaction of the analogue with the intermediary acetyl-enzyme form of the catalytic cycle [W. Plaga et al. (1988) Eur. J. Biochem. 178,. EPR spectroscopy of this system has now identified the corresponding a-phosphoryl radical as a reaction intermediate [g = 2.0032; u(P) = 2.72 mT, a(3H) = 1.96 mT]; it showed a half-life of about 20 min at 0°C. This finding proves that the enzyme radical is a hydrogen-atomtransferring coenzymic element.Pyruvate formate-lyase is the key enzyme of the anaerobic glucose degradation route in Escherichia coli cells [l]. It shows unique mechanisms of enzyme regulation and catalysis. Transcription of the structural gene (~f l )is highly controlled by anaerobic induction [2]. At the post-translational level, pyruvate formate-lyase is interconverted by specific converter enzymes between a fully inactive form (Ei) and an active form (E,) that contains a persistent organic free radical [3]. Pyruvate is nonoxidatively processed to acetyl-CoA by a two-step reaction pattern with an intermediary acetyl-enzyme:(2) Addressing structure/function relationships, we have recently mapped the covalent catalytic SH-group as the Cys-419 residue of the polypeptide chain [4]. These studies also showed that the acetyl-enzyme intermediate will react with the formate analogue hypophosphite to produce l-hydroxyethylphosphonate, linked as a thioester to Cys-418. The C-P bond-forming process, which resembles pyruvate synthesis from formate (reversal of Eqn l), has now been investigated by EPR spectroscopy. We have identified a substrate-based radical intermediate, thus demonstrating for the first time a functional role of the free radical of pyruvate formate-lyase. Our findings substantiate the notion that the reversible C-C bond cleavage/synthesis in the first half-reaction of the catalytic cycle occurs by a radical mechanism.We also report in this paper the EPR and ultraviolet spectroscopic characterization of the enzyme radical, its quantification, and several isotope substitution experiments for approachi...

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“…coli thus utilizes post-translational glycyl radical generation as part of its regulatory mechanism for two of its most central metabolic functions: glucose and nucleotide metabolism. Once generated, the glycyl radicals in PFL [18] and ARR [19] are stable under anaerobic conditions; further, the radical is not consumed during catalysis but rather is a true cofactor. Thus once the respective activating enzymes activate PFL and ARR, the glycyl radicals are present and the enzymes are active indefinitely.…”

Section: Introductionmentioning

confidence: 99%

Inactivation of E. coli pyruvate formate-lyase: Role of AdhE and small molecules

Nnyepi

Peng

Broderick

2007

Archives of Biochemistry and Biophysics

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E. coli AdhE has been reported to harbor three distinct enzymatic activities: alcohol dehydrogenase, acetaldehyde-CoA dehydrogenase, and pyruvate formate-lyase (PFL) deactivase. Herein we report on the cloning, expression, and purification of E. coli AdhE, and the re-investigation of its purported enzymatic activities. While both the alcohol dehydrogenase and acetaldehyde-CoA dehydrogenase activities were readily detectible, we were unable to obtain any evidence for catalytic deactivation of PFL by AdhE, regardless of whether the reported cofactors for deactivation (Fe(II), NAD, and CoA) were present. Our results demonstrate that AdhE is not a PFL deactivating enzyme. We have also examined the potential for deactivation of active PFL by small-molecule thiols. Both β-mercaptoethanol and dithiothreitol deactivate PFL efficiently, with the former providing quite rapid deactivation. PFL deactivated by these thiols can be reactivated, suggesting that this deactivation is non-destructive transfer of an H atom equivalent to quench the glycyl radical.

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Post-translational activation introduces a free radical into pyruvate formate-lyase.

Cited by 232 publications

References 10 publications

Purification of 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans. An iron-sulfur protein

Purification of 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans. An iron-sulfur protein

The free radical of pyruvate formate‐lyase

Inactivation of E. coli pyruvate formate-lyase: Role of AdhE and small molecules

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