The Pyruvate Formate-lyase System of Streptococcus faecalis

• Published in: The Journal of biological chemistry 1969-07, Vol.244 (13), p.3605-3612
• Main Authors: Lindmark, D G, Paolella, P, Wood, N P
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)83412-3

The enzyme exchanging formate with the carboxyl of pyruvate was purified 195-fold from extracts of Streptococcus faecalis 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 purification 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 B 12 , 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 FeSO 4 and 0.003 m 2,3-dimercaptopropanol. Tris, maleate, and imidazole 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- 14 C with the carboxyl group of oxalacetate, α-ketoglutarate, and α-ketobutyrate. The velocities relative to the formate-pyruvate exchange rate of 100% were 100%, 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 10 -5 m ), p -chloromercuribenzoate (2 x 10 -3 m ), and sodium hypophosphite (6 x 10 -6 m ) inhibit exchange activity of the crude extract to the extent of 41%, 55%, and 47%, respectively.