Pyruvate formate-lyase (inactive form) and pyruvate formate-lyase activating enzyme of Escherichia coli: isolation and structural properties.

The catalytically active form (Ea) of pyruvate formate-lyase in Escherichia coli cells is generated from an inactive form of the enzyme (Ei) through a post-translational process that requires a distinct activating enzyme and is linked to the cleavage of adenosylmethionine to methionine and 5'-deoxyadenosine. Ei and the activating enzyme were purified to homogeneity and structurally characterized. Ei has an alpha 2 oligomeric structure (2 X 85 kDa) and contains no cofactor. The amino acid composition has been determined. Out of a total of six cysteinyl residues per subunit, one shows an unusually fast reaction with iodoacetate (k2 = 7 (M-1 s-1) at pH 6.8, 30 degrees C), which is accompanied by loss of the activatability of the enzyme. The 1500-fold purified activating enzyme is a monomeric protein of 30 kDa. It contains a covalently bound, as yet unidentified chromophoric factor which has an optical absorption peak at 388 nm. Further studies of the in situ state of pyruvate formate-lyase detected a reversible backconversion of the active form Ea into Ei when anaerobic cells become nutrient-depleted.