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-

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A strain of Escherichia coli with a mutation in the ana gene was shown to lack acetaldehyde dehydrogenase and alcohol dehydrogenase. The requirement of this strain for an external oxidant to grow anaerobically on glucose shows that the reduction of acetyl-CoA is the principal means of reoxidation of NADH produced during glycolysis in E. coli. Further mutants derived from the ana strain were shown to be affected in the enzymes involved in the fermentation of pyruvate (pyruvate formate-lyase, phosphotransacetylase, acetate kinase). A gene controlling acetate kinase (ackB) activity has been located at 39 min on the chromosomal map. Evidence is presented that anaerobic nitrite reduction with pyruvate involves at least the dehydrogenase subunit of the pyruvate dehydrogenase complex.

It is shown here that a plasmid (p29) derived from the transducing phage lambda aspC2 (Christiansen and Pedersen 1981) codes for pyruvate formate-lyase. The identity of the 80 kilodaltons (kd) gene product of plasmid p29 with the pyruvate formate-lyase polypeptide was proven (i) by co-migration of the gene product expressed in the maxicell system with purified enzyme on O'Farrell gels, and (ii) by comparison of the peptide maps obtained from limited proteolysis. In vivo the 80 kd form of the enzyme was proteolytically converted to a 78 kd polypeptide. The two polypeptides (80 kd and 78 kd) and their charge isomers present in purified enzyme preparations are therefore products of a single gene. Aerobically grown cells of Escherichia coli contained a basal level of pyruvate formate-lyase which was derepressed 5- to 10-fold under anaerobiosis. Derepression also occurred during anaerobic growth on glycerol plus fumarate. Presence of plasmid p29 caused overproduction of pyruvate formatelyase, 11-fold upon anaerobic growth on glucose, 14-fold upon aerobic growth on glucose and 33-fold upon aerobic growth at the expense of D-lactate.