Pyruvate:formate lyase

Pyruvate formate-lyase (PFL) catalyzes the reversible conversion of pyruvate and CoA into acetyl-CoA and formate [36,37]  

The next step is the addition of the thiyl radical to the carbonyl carbon of pyruvate, yielding a tetrahedral oxy-radical intermediate. The calculated energy of this intermediate, relative to the free reactants, is +9.9 kcal/mol, and the barrier for its formation is calculated to 12.3 kcal/mol. The barrier for the dissociation of the radical intermediate into acetylated cysteine and formyl radical is calculated to be only 2.8 kcal/mol with an exothermicity of 3.9 kcal/mol. Taken together, the total reaction  

The localized tetrahedral intermediate of the radical pathway will readily dissociate and release formyl radical. This reactive radical is proposed by Kozarich et al to abstract a hydrogen atom from Gly734, hence regenerating the stable enzyme radical at that site. The calculations show that this proposal is perfectly feasible, having a barrier of 4.9 kcal/mol and an exothermicity of 17.5 kcal/mol. 

CoA is observed for C419S mutant, but not for C418S. Note, however, that both the suggested mechanisms are consistent with the experimental observations. 

A direct nucleophihc attack by the sulfur of non-radical cysteine on the carbonyl carbon of acetylated cysteine, yielding a tetrahedral intermediate, was also considered. As in the case of nucleophihc attack of cysteine on pyruvate, the intermediate lies relatively low in energy (+9.0 kcal/mol), but barrier is very high (41.2 kcal/mol). 

The enzyme is a homodimeric protein of A/r 170,000 and contains no known organic or metal ion cofactors. The enzyme is readily inactivated by oxygen and interconverts between active and inactive forms in vivo (173, 174). The activation process occurs under conditions of anaerobiosis and is catalyzed by an Fe(ll)-dependent activating enzyme (Mr 30,000) (775). Elegant studies on the in vitro activation of PFL by Knappe and co-workers (176, 177) have revealed that a complex activation cocktail is required, which includes the activating enzyme, pyruvate, or oxamate as allosteric effectors, S-adenosylmethionine (SAM), and flavodoxin (775) or photoreduced 5-deazariboflavin (178). A possible role for a B12 derivative in the activation or catalytic reaction for PFL is not likely in light of the observation that E. coli 113-3, a methionine/B auxotroph, pos- 

The primary structure of E. coli PFL and its activating enzyme have been determined from the DNA nucleotide sequences (184). A particular region of interest is that containing the adjacent cysteine residues at positions 418-419. Knappe and co-workers have suggested that the cysteine residue at 419 is the site of the acetylthioester formation in the catalytic cycle of the enzymic reaction (185). 

The discovery that the active form of PFL contains a paramagnetic species suggests the intriguing possibility that this unusual enzymic reaction proceeds via a homolytic chemical mechanism. Testing of this hypothesis by Kozarich and coworkers has led to studies on the inactivation of PFL by a variety of compounds 

Compounds Tested as Inactivators of Pyruvate Formate-Lyase 

Compound Concentration (mAf) Half-life (inact min) Substrate protection  

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Wagner AFV, M Frey, FA Neugebauer, W Schafer (1992) The free radical in pyruvate formate-lyase is located on glycine-734. Proc Natl Acad USA 89 996-1000. 

Knappe, J. and Sawers, G. (1990) A radical-chemical route to acetyl-CoA the anaerobically induced pyruvate formate-lyase system of Escherichia coli. FEMS Microbiology Reviews 75, 383-398. 

The realization of the widespread occurrence of amino acid radicals in enzyme catalysis is recent and has been documented in several reviews (52-61). Among the catalytically essential redox-active amino acids glycyl [e.g., anaerobic class III ribonucleotide reductase (62) and pyruvate formate lyase (63-65)], tryptophanyl [e.g., cytochrome peroxidase (66-68)], cysteinyl [class I and II ribonucleotide reductase (60)], tyrosyl [e.g., class I ribonucleotide reductase (69-71), photosystem II (72, 73), prostaglandin H synthase (74-78)], and modified tyrosyl [e.g., cytochrome c oxidase (79, 80), galactose oxidase (81), glyoxal oxidase (82)] are the most prevalent. The redox potentials of these protein residues are well within the realm of those achievable by biological oxidants. These redox enzymes have emerged as a distinct class of proteins of considerable interest and research activity. 

Studies on three different iron-sulfur enzyme systems, which all require S-adenosyl methionine—lysine 2,3-aminomutase, pyruvate formate lyase and anaerobic ribonucleotide reductase—have led to the identification of SAM as a major source of free radicals in living cells. As in the dehydratases, these systems have a [4Fe-4S] centre chelated by only three cysteines with one accessible coordination site. The cluster is active only in the reduced... 

Pyruvateiferredoxin 2-oxidoreductase, PYRUVATE SYNTHASE PYRUVATE FORMATE LYASE PYRUVATE KINASE... 

The enzymes that utilise Fe-S clusters and S-adenosylmethionine to generate radicals essential for catalysis are now identified as a class or superfamily, the radical-SAM enzymes. Hoffman et al. have studied the pyruvate formate-lyase activating enzyme (PFL-AE) by cw EPR (X-band) and pulsed ENDOR (2H and 13C, Q-band) and used the S = signals of the Fe4S4 cluster and derivatives to construct a model for the interaction of adenosylmethionine with the cluster.86... 

Boxma B, Voncken F, Jannink S, van Alen T, Akhmanova A, van Weelden SWH, van Hellemond JJ, Ricard G, Huynen M, Tielens AGM, Hackstein JHP (2004) The anaerobic chytridiomycete fungus Piromyces spE2 produces ethanol via pyruvate formate lyase and an alcohol dehydrogenase E. Mol Microbiol 51 1389-1399 Brocks JJ, Love GD, Summons RE, Knoll AH, Logan GA, Bowden SA (2005) Biomarker evidence for green and purple sulphur bacteria in a stratified Palaeoproterozoic sea. Nature 437 866-870... 

Fig. 5 Speculative metabolic schemes of the main pathways in carbohydrate metabolism in Trimyema compressum (after Goosen et al. 1990). End products are in boxes. Abbreviations AcCoA, acetyl-Co A, Hyd, hydrogenase, PEP, phosphoenolpyruvate carboxykinase, PFL, pyruvate formate lyase, PFO, pyruvate ferredoxin oxidoreductase, PYR, pyruvate, Xox, red> unknown electron carrier...

Akhmanova A, Voncken FGJ, Hosea KM, Harhangi H, Keltjens JT, den Camp HJMO, Vogels GD, Hackstein JHP (1999) A hydrogenosome with pyruvate formate-lyase anaerobic chytrid fungi use an alternative route for pyruvate catabolism. Mol Microbiol 32 1103-1114... 

Gelius-Dietrich G, Henze (2004) Pyruvate formate lyase (PFL) and PFL activating enzyme in the chytrid fungus Neocallimastix frontalis A free-radical enzyme system conserved across divergent eukaryotic lineages. J Eukaryot Microbiol 51 456-463... 

Kessler D, Herth W, Knappe J (1992) Ultrastructure and pyruvate formate-lyase radical quenching property of the multienzymatic ADHE protein of Escherichia coli. J Biol Chem 267 18073-18079... 

When grown in alkaline media, certain species of lactic acid bacteria decrease production of LDH, resulting in increased formation of formate, acetate, and ethanol as end products. This phenomenon has been observed in S. faecalis subsp. liquefaciens (Gunsalus and Niven 1942), Streptococcus durans, S. thermophilus, (Platt and Foster 1958), and Lactobacillus bulgaricus (Rhee and Pack 1980). Data of Rhee and Pack (1980) indicate that a phosphoroclastic split of pyruvate occurs under alkaline conditions to yield ATP. The enzymes involved in this reaction (pyruvate formate-lyase and acetate kinase) require alkaline conditions for optimum activity. A shift from homo- to heterofermentation because of increased pH has not been observed for Group N streptococci. 

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