Mechanism of 3-mercaptopicolinic acid inhibition of hepatic phosphoenolpyruvate carboxykinase (GTP).
The hypoglycemic agent 3-mercaptopicolinic acid inhibits gluconeogenesis from lactate by isolated, perfused livers from fasted rats and guinea pigs. A 3-mercaptopicolinate concentration of 50 muM caused a sharp decrease in glucose synthesis, with virtually complete inhibition at 100 muM. 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 consumption was not altered, even at the highest concentration of inhibitor. Gluconeogenesis from glycerol by guinea pig liver was blocked completely by 100 muM 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 bu isolated guinea pig liver mitochondria metabolizing alpha-ketoglutarate (State 3) was inhibited markedly by 3-mercaptopicolinate, but malate conversion to P-enolpyruvate was considerably less sensitive. 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 MnGTP2-, and that simulataeous 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 3 to 9 muM 3-mercaptopicolinate, and the inhibition patterns were dependent on the concentration of free Mn2+ present. Comparison of the inhibition constants 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+.
D000818:Animals; D002413:Cations, Divalent; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology; D005943:Gluconeogenesis / Q000187:drug effects; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006168:Guinea Pigs; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D008297:Male; D008345:Manganese / Q000494:pharmacology; D008433:Mathematics; D010101:Oxygen Consumption / Q000187:drug effects; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000037:antagonists & inhibitors*; D010848:Picolinic Acids / Q000494:pharmacology*; D051381:Rats; D013438:Sulfhydryl Compounds / Q000494:pharmacology
D002413:Cations, Divalent; D010848:Picolinic Acids; D013438:Sulfhydryl Compounds; D008345:Manganese; D006160:Guanosine Triphosphate; D010729:Phosphoenolpyruvate Carboxykinase (GTP)