Mechanism of 3 mercaptopicolinic acid inhibition of hepatic phosphoenolpyruvate carboxykinase (GTP)

The hypoglycemic agent 3 mercaptopicolinic acid inhibits gluconegenesis from lactate by isolated, perfused livers from fasted rats and guinea pigs. A 3 mercaptopicolinate concentration of 50 μm caused a sharp decrease in glucose synthesis, with virtually complete inhibition at 100 μm. This inhibitory effect was reversed completely when 3 mercaptopicolinate was removed and the rate of glucose synthesis returned to normal values within 2 min. Oxygen cnsumption was not altered, even at the highest concentration of inhibitor. Gluconeogenesis from glycerol by guinea pig liver was blocked completely by 100 μm 3 mercaptopicolinate but was inhibited only partially in rat liver. After removal of the inhibitor glucose synthesis returned to levels higher than noted before the addition of this compound. The formation of P enolpyruvate by isolated guinea pig liver mitochondria metabolizing α ketoglutarate (State 3) was inhibited markedly by 3 mercaptopicolinate, but malate conversion to P enolpyruvate was considerably less sensitive to inhibition. Kinetic studies with purified P enolpyruvate carboxykinase from rat liver cytosol indicate that 3 mercaptopicolinate is a noncompetitive inhibitor with respect to both oxalacetate and MnGTP^2-, and that simultaneous saturation with both substrates does not diminish this inhibition. The inhibitory effects of 3 mercaptopicolinate occur primarily by decreasing the rate of product formation while having relatively minor effects on the apparent Michaelis constants for substrates. Inhibition constants for slope and intercept effects ranged from 2 to 9 μm 3 mercaptopicolinate, and the inhibition patterns were dependent on the concentration of free Mn²⁺ present. Comparison of the inhibition constant with the observed inhibition of gluconeogenesis in livers perfused with 3 mercaptopicolinate supports the contention that P enolpyruvate carboxykinase is the site of action of this inhibitor. The possibility that 3 mercaptopicolinate inhibition occurs by binding either free or bound manganese was eliminated by determination of the dissociation constant of 0.51 mM for the manganese 3 mercaptopicolinate complex. In addition, no tightly bound, slowly exchanging metal was bound to purified enzyme protein. These results suggest that 3 mercaptopicolinate inhibits by the removal of a tightly bound, rapidly exchanging metal ion other than Mn²⁺.