Effects of vitamin C on phosphoenolpyruvate carboxykinase from rat liver.

Phosphoenolpyruvate carboxykinase (GTP) (PEPCK) is one of the key enzymes of gluconeogenesis. Mechanisms responsible for rapid regulation of enzyme activity include activation by bivalent cations such as Mn2+ and Fe2+ and/or alterations of the oxidation state of the enzyme's SH groups. A cytosolic cell free system prepared from rat liver was used to study the effects of the thiol reagents GSH and dithiothreitol (DTT) and particularly of vitamin C on PEPCK activity. (1) Basal activity and Mn(2+)-stimulated activity were not affected by variations in the concentrations of GSH and DTT, indicating that some components of the cell free system provided sufficient protection against PEPCK-inactivation due to disulfide bond formation. The latter phenomenon is known to occur with purified PEPCK in the absence of added thiols. Only in the presence of 2 microM Fe2+, GSH/DTT addition increased PEPCK activity. (2) Addition of vitamin C in the range of 0.6-1.2 mM resulted in a marked stimulation of the PEPCK reaction, ranging from 1.5-fold (with 2 microM Mn2+) to 4-7-fold (for basal activity and with 2 microM Fe2+). (3) When 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB) was used to induce disulfide bond formation and subsequent inactivation of PEPCK, reactivation experiments with GSH/DTT but not with vitamin C restored full enzyme activity. It is concluded that vitamin C activates PEPCK or protects it from inactivation caused by oxidants by a mechanism that does not involve the reduction of the enzyme's thiols.