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...
