The pyruvate formate-lyase system of Streptococcus faecalis. I. Purification and properties of the formate-pyruvate exchange enzyme.
نویسندگان
چکیده
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.
منابع مشابه
Purification of pyruvate formate-lyase from Streptococcus mutans and its regulatory properties.
Pyruvate formate-lyase (EC 2.3.1.54) from Streptococcus mutans strain JC2 was purified in an anaerobic glove box, giving a single band on disk and sodium dodecyl sulfate electrophoresis. This enzyme was immediately inactivated by exposure to the air. Enzyme activity was unstable even when stored anaerobically, but the activity was restored by preincubating the inactivated crude enzyme with S-ad...
متن کاملFormate-pyruvate Exchange Reaction in Streptococcus Faecalis. I. Factor Requirement for Intact Cells.
Wood, N. P. (A. & M. College of Texas, College Station), and D. J. O'Kane. Formate-pyruvate exchange reaction in Streptococcus faecalis. I. Factor requirement for whole cells. J. Bacteriol. 87:97-103. 1964.-A factor present in plant and animal sources was found necessary for the incorporation of formate-C(14) into pyruvate by Streptococcus faecalis 10Cl. Yeast extract produced a response linear...
متن کاملEffects of oxygen on pyruvate formate-lyase in situ and sugar metabolism of Streptococcus mutans and Streptococcus sanguis.
The strictly anaerobic metabolism of sugar in strains of Streptococcus mutans and Streptococcus sanguis was studied because deep layers of dental plaque are strictly anaerobic. Galactose-grown cells of these streptococcal strains had higher pyruvate formate-lyase activity than did glucose-grown cells. Among these strains, two strains of S. mutans had a significantly higher pyruvate formate-lyas...
متن کاملOxygen sensitivity of sugar metabolism and interconversion of pyruvate formate-lyase in intact cells of Streptococcus mutans and Streptococcus sanguis.
Pyruvate formate-lyase (PFL) (formate acetyltransferase; EC 2.3.1.54) of oral streptococci is essential for metabolizing sugar into volatile compounds (formate, acetate, and ethanol). This enzyme is extremely sensitive to oxygen, and its activity is irreversibly inactivated by oxygen. When Streptococcus sanguis was anaerobically starved, a part of the active form of PFL was converted into a rev...
متن کاملStructural basis for glycyl radical formation by pyruvate formate-lyase activating enzyme.
Pyruvate formate-lyase activating enzyme generates a stable and catalytically essential glycyl radical on G(734) of pyruvate formate-lyase via the direct, stereospecific abstraction of a hydrogen atom from pyruvate formate-lyase. The activase performs this remarkable feat by using an iron-sulfur cluster and S-adenosylmethionine (AdoMet), thus placing it among the AdoMet radical superfamily of e...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- The Journal of biological chemistry
دوره 244 13 شماره
صفحات -
تاریخ انتشار 1969