The pyruvate formate-lyase system of Streptococcus faecalis. I. Purification and properties of the formate-pyruvate exchange enzyme.

The enzyme exchanging formate with the carboxyl of pyruvate was purified 195-fold from extracts of Streptococcus fuecalis by a procedure involving protamine treatment, dialysis, and gradient centrifugation. Centrifugation of protamine-treated extract in a 10 to 40% potassium tartrate gradient provided a 65-fold purhication and separated the exchange enzyme from pyruvate decarboxylase, acetolactate synthetase, acetolactate decarboxylase, and phosphate-acetyltransferase. A second centrifugation in a 20 to 30% potassium tartrate gradient provided an additional 3-fold purification. The purified preparation contains diphosphothiamine but not coenzyme A, folate, lipoate, biotin, pyridoxal, pyridoxamine, Vitamin Blz, or flavin. The enzyme has an average molecular weight of 268,000 to 288,000 as estimated by gradient centrifugation and by gel filtration. Purified preparations do not degrade pyruvate. S-Adenosyl-L-methionine has no effect on the purified enzyme; the apparent stimulation of activity with crude extracts apparently results from inhibition of competing reactions. The purified enzyme is rapidly oxidized and inactivated, but it can be stabilized in 30% potassium tartrate containing a reducing complex of 0.001 M FeS04 and 0.003 M 2,3-dimercaptopropanol. Tris, maleate, and hnidazole acetate buffers will substitute for phosphate buffer and phosphate ion is not required for the reaction. Optimal activity is observed over a range between pH 6.7 and 8.7. Purified extracts will exchange formate-14C with the carboxyl group of oxalacetate, ar-ketoglutarate, and cu-ketobutyrate. The velocities relative to the formate-pyruvate exchange rate of 100% were loo%, 9.9%, and 0.9%, respectively. Purified extracts give Michaelis constants of 0.02 M for pyruvate and 0.058 M for formate. Chloride ion produces an irreversible inhibition of exchange activity. Arsenite (3 X 1O-5 M), p-chloromercuribenzoate (2 X 10va M), and sodium hypophosphite (6 X lop6 M) inhibit exchange activity of the crude extract to the extent of 41%, 55%, and 47%, respectively.