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Succinic Thiokinase

Methods for the preparation of guanosine triphosphate-γ- 32 P, 3 H-coenzyme A, and 14 C-succinyl- 3 H-CoA are described. Various forms of succinic thiokinase from pig heart are prepared and isolated. They include the phosphorylated enzyme, the enzyme-bound CoA, and the enzyme-bound succinyl-CoA. Fr...

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Bibliographic Details
Published in:The Journal of biological chemistry 1967-06, Vol.242 (11), p.2582-2592
Main Authors: Cha, Sungman, Cha, Chung-Ja Mo, Parks, R.E.
Format: Article
Language:English
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Summary:Methods for the preparation of guanosine triphosphate-γ- 32 P, 3 H-coenzyme A, and 14 C-succinyl- 3 H-CoA are described. Various forms of succinic thiokinase from pig heart are prepared and isolated. They include the phosphorylated enzyme, the enzyme-bound CoA, and the enzyme-bound succinyl-CoA. From the amounts of those substrates bound to the enzyme, it is concluded that a molecule of the enzyme has at least two, and probably four, binding sites (presumably also catalytic centers). The enzyme catalyzes the 14 C-GDP exchange with GTP requiring Mg ++ , 32 P-inorganic phosphate exchange with GTP requiring Mg ++ and CoA, and the exchanges of 14 C-succinate and 3 H-CoA with succinyl-CoA in the presence of Mg ++ ions. These isotope exchanges and the occurrence of various intermediates are in general agreement with the previously suggested reaction mechanism consisting of three partial reactions and two high energy forms of the enzyme, succinic thiokinase ∼ P and succinic thiokinase ∼ CoA. However, a number of considerations are presented which must be clarified further before a definitive conclusion is reached. Incidental to the succinic thiokinase work, the occurrence of a phosphorylated intermediate of nucleoside diphosphokinase from pig heart has been found.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(18)99612-2