Succinic thiokinase of Escherichia coli. Purification, phosphorylation of the enzyme, and exchange reactions catalyzed by the enzyme

Frederick Lawrence Grinnell, Jonathan S. Nishimura

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Abstract

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 DEAE-cellulose chromatography and gel filtration on Sephadex G-150. Assuming a molecular weight of 141,000 (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 non-phosphorylated 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.

Original languageEnglish (US)
Pages (from-to)562-568
Number of pages7
JournalBiochemistry
Volume8
Issue number2
StatePublished - 1969

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Succinate-CoA Ligases
Phosphorylation
Escherichia coli
Purification
Ion exchange
Coenzyme A
Enzymes
Adenosine Triphosphate
Succinic Acid
Adenosine Diphosphate
Phosphates
Coenzyme A Ligases
DEAE-Cellulose
Enzyme activity
DEAE-Cellulose Chromatography
Chromatography
Adenine Nucleotides
Gels
Molecular weight
Gel Chromatography

ASJC Scopus subject areas

  • Biochemistry

Cite this

Succinic thiokinase of Escherichia coli. Purification, phosphorylation of the enzyme, and exchange reactions catalyzed by the enzyme. / Grinnell, Frederick Lawrence; Nishimura, Jonathan S.

In: Biochemistry, Vol. 8, No. 2, 1969, p. 562-568.

Research output: Contribution to journalArticle

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