Pyruvate formate-lyase (acetyl-CoA:formate C-acetyltransferase, EC 2.3.1.54) from anaerobic Escherchia coli cells converts pyruvate to acetyl-CoA and formate by a unique homolytic mechanism that involves a free radical harbored in the protein structure. By EPR spectroscopy of selectively 13C-labeled enzyme, the radical (g = 2.0037) has been assigned to carbon-2 of a glycine residue. Estimated hyperfine coupling constants to the central 13C nucleus (Au = 4.9 mT and A, = 0.1 mT) and to 13C nuclei in a and 13 positions agree with literature data for glycine radical models. N-coupling was verified through uniform I'N-labeling. The large IH hyperfine splitting (1.5 mT) dominating the EPR spectrum was asignd to the a proton, which in the enzyme radical is readily solvent-exchangeable. 996The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Pyruvate formate-lyase (formate acetyltransferase; EC 2.3.1.54) of Escherichia coli cells is post-translationally interconverted between inactive and active forms. Conversion of the inactive to the active form is catalyzed by an Fe2 -dependent activating enzyme and requires adenosylmethionine and dihydroflavodoxin. This process is shown here to introduce a paramagnetic moiety into the structure of pyruvate formate-lyase. It displays an EPR signal at g = 2 with a doublet splitting of 1.5 mT and could comprise an organic free radical located on an amino acid residue of the polypeptide chain. Hypophosphite was discovered as a specific reagent that destroys both the enzyme radical and the enzyme activity; it becomes covalently bound to the protein. (Fl). The latter is generated from NADPH-and pyruvate-dependent oxidoreductases (4). Studies of the reconstituted system have suggested that the conversion reaction proceeds as follows (5) 1332The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

The reaction pyruvate + CoA % acetyl-CoA + formate, catalyzed by pyruvate formate-lyase of Escherichia coli, occurs by the succeeding half-reactions (a) E + pyruvate e E-acetyl + formate; (b) E-acetyl + CoA Making use of coupled optical assays, a 'ping-pong mechanism' was derived from the complete kinetic investigation of the forward and reverse reactions. The thermodynamic equilibrium constant of the overall reaction was calculated from the kinetic constants to be K = 750 (30 "C, pH 8.1), which agrees with chemically determined values.The intermediate acetyl-enzyme, which had been previously proposed from the ['4C]formatepyruvate exchange, was detected by product-pulse experiments with [2-'4C]pyruvate and trapped by acid precipitation. The acetyl group is linked to a sulfhydryl group of the protein.The value of the equilibrium constant of the first half-reaction is about 50, as directly measured and calculated from the kinetic data. It was concluded that the standard free energy of hydrolysis of acetyl-enzyme is about 1.7 kcal (7.1 kJ) more negative than that of acetyl-CoA.The intermediate was found to react with dithiothreitol with a second-order rate constant at 30°C and pH 7.6 of 1160 M-' xmin-'. It resulted in a half-life of 4 s (or 20 s at 0°C) in the particular buffer which was required for enzyme stabilization.The enzyme (about 60 U/mg) was prepared by carrying its purified inactive form through the enzyme-11-dependent activation reaction, employing photoreduced flavodoxin along with the effector compounds S-adenosylmethionine and oxamate (as a pyruvate analogue).The conversion of pyruvate into acetyl-CoA according to the equation Pyruvate + CoASH e Acetyl-SCoA + formate (1) plays a key role in the metabolism of Escherichia coli. It is the anaerobic counterpart of pyruvate oxidation by the pyruvate dehydrogenase complex and is operative alternatively to the latter [1,2].Being a unique type of reaction of a 2-oxocarbonic acid, the mechanism of the pyruvate formate-lyase Abbreviation. Mops, 2-(N-morpholino)propanesulfonate. Enzymes (CBN recommendations 1972). Pyruvate for-

Anaerobically growing Escherichia coli cells contain the enzyme pyruvate formate‐lyase which catalyses the non‐oxidative cleavage of pyruvate to acetyl‐CaA and formate. The enzyme is subject to interconversion between inactive and active forms. The active form contains an oxygen‐sensitive organic free radical located on the polypeptide chain which is essential for catalysis. It affords a novel homolytic C‐C bond cleavage of the pyruvate substrate. The radical is generated by an iron‐dependent converter enzyme which requires reduced flavodoxin and adenosyl methionine as co‐substrates and pyruvate as a positive allosteric effector. A second converter enzyme, also iron‐dependent, accomplishes the removal of the radical. This post‐translational interconversion cycle controls the activity state of pyruvate formate‐lyase in the anaerotic cell. Anaerobic conditions also regulate pyruvate formate‐lyase at the level of gene expression. Multiple promoters are responsible for effecting a twelve to fifteen fold induction and they are coordinately controlled in response to the oxygen and metabolic status of the cell by sequences which are located far upstream of the pfl coding region. The transcription factor Fnr has been identified as being responsible for part of the anaerobic control of pfl expression, probably through direct interaction with the upstream sequences. In contrast, the expression of the gene encoding the first iron‐dependent converter enzyme is unaffected by anaerobiosis and is independent of the Fnr protein.

A 4.8 kb DNA‐fragment was cloned and sequenced encompassing the structural gene of PFL‐deactivase (2.7 kb) and 2 kb of the 5 flanking region that contains the elements for anaerobic induction. A mutant lacking deactivase was shown to require exogenous electron acceptors for anaecrobic growth with glucose. This revealed the identity of PFL‐deactivase with the alcohol and acetaldehyde dehydrogenases of E. coli. The multienzyme represents a homopolymeric protein (∼ 40 × 96 kDa) requiring Fe2+ for all functions.

The glycyl radical (Gly-734) contained in the active form of pyruvate formate-lyase (PFL) of Escherichia coli is generated by the S-adenosylmethionine-dependent pyruvate formate-lyase-activating enzyme (PFL activase). A 5-deoxyadenosyl radical intermediate produced by the activase has been suggested as the species that abstracts the pro-S hydrogen of the glycine 734 residue in PFL (Frey, M., Rothe, M., Wagner, A. F. V., and Knappe, J. (1994) J. Biol. Chem. 269, 12432-12437). To enable mechanistic investigations of this system we have worked out a convenient large scale preparation of functionally competent PFL activase from its apoform. The previously inferred metallic cofactor was identified as redox-interconvertible polynuclear iron-sulfur cluster, most probably of the [4Fe-4S] type, according to UV-visible and EPR spectroscopic information. Cys 3Ser replacements by site-directed mutagenesis determined Cys-29, Cys-33, and Cys-36 to be essential to yield active holoenzyme. Gel filtration chromatography showed a monomeric structure (28 kDa) for both the apoenzyme and holoenzyme form. The iron-sulfur cluster complement proved to be a prerequisite for effective binding of adenosylmethionine, which induces a characteristic shift of the EPR signal shape of the reduced enzyme form ([4Fe-4S] ؉ ) from axial to rhombic symmetry.Pyruvate formate-lyase (PFL) 1 is a key enzyme of the anaerobic glucose fermentation in Escherichia coli and other microorganisms, catalyzing the CoA-dependent cleavage of pyruvate to acetyl-CoA and formate (1). In its active form, this enzyme contains a stable glycyl radical (Gly-734) required for catalysis (2). Studies of mutants and substrate analogs propose that on substrate binding, the spin is transferred from Gly-734 to the reaction center (Cys-418/Cys-419), where a thiyl radical initiates the homolytic cleavage of the pyruvate C-C bond (3, 4).The PFL radical is produced post-translationally by abstraction of the H si atom from the Gly-734 methylene group (5). This occurs by the action of pyruvate formate-lyase-activating enzyme (PFL activase), which employs adenosylmethionine (AdoMet) and dihydroflavodoxin (or artificial 1eϪ donors) as co-substrates, yielding 5Ј-deoxyadenosine and methionine as stoichiometric co-products (Equation 1).Because the abstracted H atom is recovered in the 5Ј-CH 3 of deoxyadenosine, a 5Ј-deoxyadenosyl radical intermediate has been proposed as the actual H abstracting species in this system. How this nucleoside radical is generated from AdoMet is an intriguing problem, requiring in the first place PFL activase to be fully characterized at the molecular level. Previous work has identified PFL activase as a monomer of 28 kDa, and its primary structure, as deduced from the DNAnucleotide sequence (246 amino acids), has been established by N-and C-terminal amino acid sequencing (6, 7). The enzyme has long since been recognized to require Fe 2ϩ for catalytic activity (6), and iron contents in the order of one iron/polypeptide chain were determined in purified prepara...

Pyruvate formate-lyase (also called formate acetyltransferase; EC 2.3.1.54; PFI .) catalyses the thiolytic cleavage of pyruvate by CoA, yielding acetyl-CoA and formate. This reaction is the key step in the glucose-fermentation route in Escherichziz coli and various other bacteria. Operationally, it resembles the (B-keto)thiolase reaction of the fatty-acid degradation cycle. The mechanism of pyruvate formate-lyase, however, is fundamentally different, since the carbon-carbon bond of its substrate is cleaved homolytically rather than heterolytically. This property emerged with the discovery of a protein-based radical in the active enzyme form [ 11. The unpaired spin has recently been assigned to C-2 of (;lyi" [ 2 ] .The radical is produced by a postribosomal hydrogen-atom abstraction that is catalysed by PFI, activase using adenosylmethionine (AdoMet) and reduced flavodoxin as co-substrates [ 11. A separate reaction that quenches the protein radical in PFI, is catalysed by the multifunctional AdhE protein and is initiated when anaerobic cells are shifted to positive redox potentials [3].Metabolic aspects of PFI, interconversion between inactive (E) and active (Em) forms and the genetic/transcriptional background of the system have already been reviewed 141. This review will focus on enzyme-catalytic structure/function properties.

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