105] Pyruvate formate-lyase from Escherichia coli and its activation system

Abstract

This chapter describes the assay method, purification procedure, and properties of pyruvate formate-lyase from Escherichia coli and its activation system. Pyruvate formate-lyase activity may be assayed by measuring the production of formate in Reaction (1); CoA is expediently regenerated by the phosphate acetyltransferase reaction. An optical assay, allowing the continuous recording of pyruvate formate-lyase activity, results from the coupling of the production of acetyl-CoA to the reduction of NAD by the addition of malate, malate dehydrogenase, and citrate synthase. The activation process (2) is followed by measuring the production of pyruvate formate-lyase activity, and by this means its protein components pyruvate formate-lyase (inactive) and enzyme II are assayed. The purification procedure is a slight modification of that described by Knappc et al; pyruvate formate-lyase (inactive form), enzyme II, and flavodoxin are obtained from one batch. All steps are performed at about 4°. The protein fractions of intermediate stages, free of ammonium sulfate, can be stored in the frozen state for at least 1 month. pH measurements are at 20°. The molecular weight of the inactive species of pyruvate formatelyase is 140,000; it comprises two polypeptide chains of MW about 70,000. The active form has the same size, as estimated from sedimentation velocity and gel filtration data. Pyruvate formate-lyase activity is maximal from pH 7.8 to 8.4. Activation of pyruvate formate-lyase (inactive form) requires reducing systems affording potentials of at least –0.39 V and is maximal only at –0.44 V. The anaerobic gel filtration of activated pyruvate formate-lyase is also described in the chapter.