Succinate Thiokinase

• Published in: The Journal of biological chemistry 1973-01, Vol.248 (1), p.15-24
• Main Authors: Baccanari, David P., Cha, Sungman
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)44439-6

The techniques of isoelectric focusing and polyacrylamide gel electrophoresis have been used to demonstrate multiple forms of pig heart succinate thiokinase. There are at least five peaks of enzyme having isoelectric points of 6.4, 6.2, 6.0, 5.9, and 5.8; all are similar with respect to apparent molecular weight, heat stability, pH optimum, and reaction rates with substrate analogs. Also, two other forms with isoelectric points of 5.6 and 5.3 are seen under certain conditions. Isotopic studies show that a major portion of the enzyme is phosphorylated in its native state, with the pI 6.2, 6.0, 5.9, and 5.8 peaks containing the same number of phosphates bound per unit of enzymic activity. The pI 6.4 form lacks exchangeable phosphate but can be converted to the pI 6.2, 6.0, and 5.9 forms by GTP. The pI 6.2 enzyme is converted to the 6.0 and 5.9 forms by the same treatment. When the pI 6.2 or 6.0 forms are incubated with coenzyme A, the pI 6.4 enzyme can be regenerated. Since the pI 6.4 enzyme contains neither exchangeable phosphate nor CoA, it has been called the free enzyme form. The free enzyme is unstable, losing up to 50% of its activity in 2 hours, but it may be protected by glycerol. The interconvertibility of the various enzymes is also seen when the pI 5.9 peak distributes to the other phosphorylated forms upon refocusing.