The free radical of pyruvate formate‐lyase

Abstract

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...

Microbial conversion of choline to trimethylamine requires a glycyl radical enzyme

“…The characteristic signal of a glycine-centered radical was observed only for cells grown on choline. The g value (2.003) and strong hyperfine coupling to a single proton (1.3 mT) indicate the presence of an organic radical located at the α-carbon of an amino acid and are consistent with previously reported EPR spectra of cells expressing glycyl radical enzymes (30,31). The weaker nonhyperfine isotropic signal observed for cells from pyruvate cultures likely originates from flavoproteins, which are produced by a related Desulfovibrio strain during growth on pyruvate (32).…”

Physiological Characterization of a Heme-Deficient Mutant of Staphylococcus aureus by a Proteomic Approach

“…In Escherichia coli, the pyruvate formate lyase acts as a homodimer and is activated by introduction of a free radical to a glycine residue in one subunit of the enzyme. In the presence of oxygen (for instance, during aerobic protein extraction), the radicalization (30). Thus, activated Pfl in E. coli appears in two bands on one-dimensional protein gels; these bands represent the mature form and the cleaved form.…”

Synthesis and Posttranslational Regulation of Pyruvate Formate-Lyase in Lactococcus lactis

“…As mentioned previously, E. coli PFL is activated by introduction of a free radical to a glycine residue (30) in one of the subunits of a PFL homodimer (29). The PFL subunit containing the free radical is cleaved when it is exposed to oxygen.…”

“…Therefore, with a known radical stoichiometry of one per homodimer (29), the relative intensities of the two bands reflect the fraction of PFL existing in its active form. A PFL pool consisting of 100% active PFL will after complete irreversible inactivation result in approximately equal intensities for the two bands, if the small difference in the molecular weights of the cleaved and full-length subunits does not affect antibody recognition.…”