SUCCINIC THIOKINASE OF ESCHERICHIA COLI . PURIFICATION PHOSPHORYLATION OF ENZYME AND EXCHANGE REACTIONS CATALYZED BY ENZYME

Succinic thiokinase (succinate:coenzyme A ligase (adenosine diphosphate, EC 6.2.1.5) has been isolated in highly purified form from Escherichia coli (ATCC 4157) by a procedure which includes DEAEcellulose chromatography and gel filtration on Sephadex G-150. Assuming a molecular weight of141,000 (Ramaley, R. F., Bridger, W. A., Moyer, R. W., and Boyer, P. D. (1967), J. Biol. Chem. 242 (4287), close to two phosphoryl groups are incorporated per mole of enzyme, whether the phosphorylating agent is adenosine triphosphate or inorganic phosphate (in the presence of succinyl-coenzyme A). Capability for phosphorylation appears to be related to enzyme activity and extent of phosphorylation by adenosine triphosphate is not significantly affected by coenzyme A at 2.5 × 10-5 m. At this concentration coenzyme A strongly stimulates the adenosine triphosphate ⇄ adenosine diphosphate exchange reaction catalyzed by the enzyme. It has been concluded that under these conditions coenzyme A is not bound covalently or involved in a high-energy nonphosphorylated form of the enzyme. It has also been found that inorganic phosphate is an almost complete requirement for the succinate ⇄ succinyl coenzyme A exchange reaction catalyzed by the enzyme, adding support to the hypothesis that enzyme-bound succinyl phosphate is an intermediate in the over-all reaction. © 1969, American Chemical Society. All rights reserved.