Post-translational activation introduces a free radical into pyruvate formate-lyase.
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. The enzymatic generation of the radical, which is linked to adenosylmethionine cleavage into 5'-deoxyadenosine and methionine, possibly occurs through an Fe-adenosyl complex. These results suggest a radical mechanism for the catalytic cycle of pyruvate formate-lyase.
D000123:Acetyltransferases / Q000235:genetics*; D002250:Carbon Radioisotopes; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005609:Free Radicals; D007700:Kinetics; D010721:Phosphinic Acids / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D011499:Protein Processing, Post-Translational; D012436:S-Adenosylmethionine / Q000378:metabolism; D014316:Tritium
D002250:Carbon Radioisotopes; D005609:Free Radicals; D010721:Phosphinic Acids; D010761:Phosphorus Radioisotopes; D014316:Tritium; D012436:S-Adenosylmethionine; C009287:sodium hypophosphite; D000123:Acetyltransferases; C022560:formate C-acetyltransferase